Method for preventing and treating lipid metabolism disorders and related diseases thereof

ABSTRACT

The present invention relates to a method for preventing and/or treating a fat metabolism disorder and its related conditions, comprising administering an effective amount of plasminogen to a subject susceptible to or suffering from a fat metabolism disorder and its related conditions, to reduce an abnormal fat deposition at various sites of the body, thereby achieving the purpose of preventing and/or treating a fat metabolism disorder and its related conditions or complications.

TECHNICAL FIELD

The present invention relates to a method for preventing and/or treatinga fat metabolism disorder and its related conditions, comprisingadministering an effective amount of plasminogen to a subjectsusceptible to or suffering from a fat metabolism disorder and itsrelated conditions, to reduce an abnormal fat deposition in a bodytissue and an organ, thereby achieving the purpose of preventing and/ortreating a fat metabolism disorder and its related conditions andcomplications.

BACKGROUND ART

The fat metabolism disorder, also known as lipodystrophy, is one ofmetabolic diseases. It is the abnormality in lipids and lipidmetabolites and the amounts thereof in blood and other tissues andorgans, caused by primary or acquired factors. Lipid metabolism involveslipids being digested and absorbed in the small intestine, entering theblood circulation via the lymphatic system (via lipoprotein transport),being transformed by the liver, stored in adipose tissues, and beingused by tissues when needed. The main function of lipids in the body isto provide energy through oxidation. The adipose tissue is the body'senergy store. Fat can also protect the internal organs in cooperationwith the skin, bones, and muscles, prevent body temperature loss, andhelp the absorption of fat-soluble vitamins in food. Phospholipid is animportant structural component of all cell membranes. Cholesterol is theprecursor of cholic acid and steroid hormones (adrenal cortical hormoneand gonadal hormone). Lipid metabolism is regulated by genetics,neurohumor, hormones, enzymes, and tissues and organs such as the liver.When these factors have any abnormalities, it may cause a lipidmetabolism disorder and pathophysiological changes of relevant organs,e.g., hyperlipoproteinemia and its resulting clinical syndrome, obesity,fatty liver, etc.

Hyperlipoproteinemia is caused by excessive lipoproteins in blood.Lipids in blood, e.g., triglyceride (TG), free cholesterol (FC),cholesteryl ester (CE) and phospholipid, are rarely soluble in water.Only combined with apolipoproteins (APOs) to form a giant moleculecomplex (lipoprotein), can these lipids be dissolved, transported andmetabolized in blood. Hyperlipemia occurs when blood lipids are abovethe upper limit in normal people. Hyperlipemia is also calledhyperlipoproteinemia since blood lipids are transported in the form oflipoproteins in blood. The general criteria are as follows: fastingblood triglycerides and cholesterol in adults exceed 160 mg/dl and 260mg/dl, respectively; and cholesterol in children exceeds 160mg/dl^([1]).

Hyperlipoproteinemia (hyperlipemia) is one of the important causes ofatherosclerotic lesions and is a manifestation of abnormal lipidmetabolism in the body. Due to the different types of blood lipids orlipoproteins, the types of blood lipids or lipoproteins of which thecontents are beyond the normal range may also be different. Therefore,the World Health Organization (WHO) divides hyperlipoproteinemia intofive types: Type I, mainly characterized by an increase in chylomicrons,and opalescent, turbid serum with a high amount of triglycerides (TGs);Type II, which is divided into two subtypes, IIa and IIb, wherein theformer is mainly characterized by a significant increase in low-densitylipoproteins (LDLs), and the latter is additionally characterized by anincrease in very low-density lipoproteins (VLDLs); Type III,characterized by usually turbid serum, an increase in both LDLs andVLDLs, and fusion of the two on the electrophoresis; Type IV, mainlycharacterized by an increase in VLDLs, and possibly turbid serum; andType V, characterized by increase in both chylomicrons and VLDLs, andopalescent, turbid serum. Type II and Type IV are the most common^([1]).

Hyperlipemia can be divided into two categories: primary and secondary,according to the etiology. Primary hyperlipemia is mostly caused bycongenital defects (or genetic defects) in lipid and lipoproteinmetabolisms and by some environmental factors (comprising diets,nutrition, drugs, etc.) through unknown mechanisms. Secondaryhyperlipemia is substantially secondary to certain diseases, such asdiabetes mellitus, a liver disease, a kidney disease, a thyroid disease,as well as drinking and obesity. Environmental factors such as diets andlifestyle also contribute to the disease.

Since diabetes mellitus is often associated with a lipid metabolismdisorder, diabetes mellitus is also known as “diabetesmellipitus”^([2]). The pathogenesis of diabetes mellitus is related to Bcell dysfunction and insulin resistance, presenting as chronichyperglycemia, and a disorder of glucose metabolism is often associatedwith a disorder of lipid metabolism. The lipid metabolism disorder withdiabetes mellitus has become an independent risk factor for acardiovascular disease, which is substantially manifested byhypertriglyceridemia, a low HDL level, and an increased LDLconcentration.

The pathogenesis of the lipid metabolism disorder with diabetes mellitusis still unclear, but numerous evidences show that insulin resistance isthe central link of its occurrence. Recent studies have also found thatintestinal insulin resistance is also involved. Studies in animal modelsand populations of diabetes mellitus have shown that abnormalities inthe expression of certain genes associated with lipid metabolism furthercontribute to insulin resistance. The occurrence of atherosclerosis indiabetic patients is related to various factors, but an abnormality inplasma lipid level is the most important factor. Studies have shown thatthe morbidity and mortality of cardiovascular diseases in diabeticpatients are significantly higher than those in non-diabetic patients,and that diabetes mellitus has become an independent risk factor forcardiovascular diseases^([3]).

In recent years, the relationship between nephropathy and lipidmetabolism disorders has attracted more and more attention. A chronicprogressive renal injury is often accompanied by abnormal lipidmetabolism, and in turn hyperlipemia can promote and aggravate the renalinjury, and besides mediating glomerular injury, it also plays a role ina tubulointerstitial injury. Munk first described dyslipidemia innephrotic syndrome in 1913. Some scholars have reported thathyperlipemia may appear in 70%-10% of patients with nephrotic syndrome.It is mainly manifested by a significant increase in blood totalcholesterol (TC) dominated by an increase in low-density lipoproteincholesterol; and a slight increase in triglyceride (TG), wherein theincrease in low-density lipoprotein (LDL) is correlated with urineprotein^([4]). A patient with chronic renal insufficiency is mainlymanifested by moderate triglyceridemia, generally normal plasma totalcholesterol level, increased cholesterol in VLDLC andintermediate-density lipoprotein cholesterol (IDLC), decreasedhigh-density lipoprotein cholesterol (HDLC), and increased content oftriglyceride in various lipoproteins. The underlying cause is that theuremic environment has adverse effects on the synthesis and catabolismof triglycerides and an inhibitory effect on the reverse transport ofcholesterol^([5]).

With the popularization of kidney transplantation therapy and the wideapplication of various new immunosuppressive agents (particularly CsAand prednisone), the survival period of patients with chronic renalfailure (CRF) has been significantly prolonged, but the incidence ofhyperlipemia after kidney transplantation is very high. The mainmanifestations of hyperlipemia after kidney transplantation are elevatedlevels of plasma total cholesterol (TC), triglyceride (TG), low-densitylipoprotein cholesterol (LDLC), and very low-density lipoproteincholesterol (VLDLC)^([6]).

Clinical studies have confirmed that there is a certain correlationbetween lipid metabolism disorders and diabetic nephropathy. In adiabetic patient with a lipid metabolism disorder, an elevated lipiddeposition on a glomerular basement membrane stimulates basementmembrane cell proliferation and extracellular matrix formation. As earlyas in 1936, Kimmelstiel and Wilson found massive lipid depositions inrenal arterioles, glomeruli and renal tubules of patients with diabeticnephropathy^([7]). Abnormal lipid metabolism leading to glomerular andtubulointerstitial fibrosis is one of the most important causes ofprogressive renal impairment^([8]).

Lipid metabolism disorders can also result in occurrence of obesity(obesity syndrome). Obesity is divided into two categories: simple andsecondary. Simple obesity refers to obesity without obvious endocrineand metabolic diseases, which can be divided into two types:constitutional obesity and acquired obesity. Constitutional obesity hasa family heredity history, patients have been fed with abundant foodsince childhood, with excess intake, obese since childhood, withhyperplasia and hypertrophy of adipocytes. Acquired obesity is mostlycaused by excessive nutrition and/or reduced physical activity, such ascaused by the improvement of material conditions of life after middleage, recovery from diseases and full recuperation, and the cessation ofphysical exercise or physical labor after giving birth; and adiposecells shows hypertrophy change, without hyperplasia, and the therapeuticeffect for this type of obesity is better. Secondary obesity is mainlycaused by neuroendocrine diseases. Neuroendocrine plays an importantrole in regulating metabolism: (1) Hypothalamus has the center thatregulates appetite; and the sequela of central nervous systeminflammation, trauma, tumor and the like can cause hypothalamicdysfunction, making appetite enormous and leading to obesity. (2)Insulin secretion is increased, e.g., hyperinsulinemia is caused byexcessive insulin injection in a patient with earlynon-insulin-dependent diabetes mellitus, and islet B cell tumor secretesexcessive insulin, both of which increases fat synthesis, therebycausing obesity. (3) In the case of hypopituitarism, particularly whengonadotrophin and thyrotrophin reduction causes hypogonadism andhypothyroidism, obesity may occur. (4) Multiparas or those orally takingcontraceptives for female are predisposed to obesity, suggesting thatoestrogen has a role in promoting fat synthesis. (5) Hypercortisolism isoften accompanied by centripetal obesity. (6) Hypothyroidism with a lowmetabolic rate leads to fat accumulation with myxedema. (7) Hypogonadismmay also lead to obesity, such as dystrophia adiposogenitalis (alsonamed cerebral adiposity and Frohlich's syndrome, caused by trauma,encephalitis, pituitary tumor, craniopharyngioma and other injuries inthe hypothalamus, manifested as centripetal obesity with diabetesinsipidus and sexual retardation).

Lipid metabolism disorders often lead to fatty liver. Fatty liver refersto a lesion caused by excessive fat accumulation in liver cells due tovarious reasons. The liver plays a particularly important role in lipidmetabolism, it synthesizes lipoproteins which facilitates lipidtransport, and is also a major site for fatty acid oxidation and ketonebody formation. The normal content of lipid in liver is not much, about4%, substantially comprising phospholipid. If the liver cannot transportfat out in time, fat accumulates in the liver cells, thereby formingfatty liver.

Fatty liver can be an independent disease or can be caused by othercauses, such as obesity-induced fatty liver, alcoholic fatty liver,rapid weight loss induced fatty liver, malnutrition-induced fatty liver,diabetic fatty liver, drug-induced fatty liver, etc.

Fatty liver may be caused by inhibition of the synthesis of proteins bysome drugs or chemical poisons such as tetracycline, adrenocorticalhormone, puromycin, cyclohexylamine, emetine, arsenic, lead, silver, andmercury. Hypolipidemic drugs can also result in fatty liver byinterfering with lipoprotein metabolism.

One of the hazards of fatty liver is that it promotes the formation ofatherosclerosis. One of the causes of atherosclerosis is that a patientwith fatty liver is often accompanied by hyperlipemia, and thus bloodviscosity is increased, wherein low-density lipoprotein (LDL) can easilypenetrate an arterial intima and deposit on a vascular wall due to itsextremely small molecular weight, which reduces the arterial elasticity,narrows the vascular diameter, weakens the flexibility, and finallyleads to the disturbance of blood circulation. The second hazard offatty liver is to induce or aggravate hypertension, and coronary heartdisease, and easily lead to myocardial infarction and thus sudden death.The third hazard of fatty liver is encephalopathy-liver fattymetamorphosis syndrome (Reye's syndrome). The fourth hazard of fattyliver is to lead to hepatic cirrhosis, liver failure, and liver cancer.

Fatty liver is the product of a lipid metabolism disorder in liver andalso the pathogenic factor that aggravates liver injury, which is adevelopment of mutual causation and vicious circle. The lipid dropletsin the hepatocytes are increased, resulting in steatosis and enlargementof the hepatocytes, and extrusion of the nuclei away from the center.Fat metabolism mainly takes place in the mitochondria. Fat istransported out of the cell mainly through the smooth endoplasmicreticulum. Fat accumulation in hepatocytes further aggravates the burdenof mitochondria and endoplasmic reticulum and reduces their functions,thus affecting the metabolism of other nutrients, hormones and vitamins.Long-term hepatocyte degeneration will lead to regeneration disorder andnecrosis of hepatocytes, and thus form liver fibrosis and hepaticcirrhosis. The incidence of hepatocellular carcinoma secondary tohepatic cirrhosis is higher.

The fifth hazard of fatty liver is acute gestational fatty liver with ahigh mortality. The disease, also known as obstetric acute yellowhepatatrophia, is a rare pregnancy complication with a bad prognosis.The disease occurs mostly in the last three months of pregnancy, and itsclinical manifestations are often similar to acute severe liver disease,and comprise acute liver failure, pancreatitis, renal failure, andsystemic coagulation abnormality, leading to rapid death. The diseaseoccurs mostly in pregnant women who are pregnant for the first time.

The sixth hazard of fatty liver is to induce or aggravate diabetesmellitus. If the concentration of blood glucose in a patient withobesity-induced fatty liver exceeds the normal level, generallypre-diabetes mellitus is considered true although this situation doesnot meet the diagnostic criteria of diabetes mellitus. Fatty liver anddiabetes mellitus often accompany each other and interact with eachother, which brings greater difficulties to clinical treatment.

The studies of the present invention found that plasminogen can preventand/or reduce an abnormal fat deposition in a body tissue and an organ,for instance, it can prevent and reduce an abnormal lipid deposition inblood, a vascular wall, an internal organ, and a tissue between organs,and improve the function of these tissues and organs, thus providing anew preventive and therapeutic solution for a fat metabolism disorderand its related conditions, as well as the accompanying diseases orcomplications.

SUMMARY OF THE INVENTION

The present invention relates to the prevention and/or treatment of afat metabolism disorder and its related conditions in a subject.

In one aspect, the present invention relates to a method for preventingand/or treating a fat metabolism disorder and its related conditions ina subject, comprising administering a prophylactically and/ortherapeutically effective amount of plasminogen to the subject, whereinthe subject is susceptible to a fat metabolism disorder, suffers from afat metabolism disorder or other diseases accompanied by a fatmetabolism disorder. The present invention further relates to the use ofplasminogen for preventing and/or treating a fat metabolism disorder andits related conditions in a subject. The present invention furtherrelates to the use of plasminogen in the preparation of a medicament, apharmaceutical composition, an article of manufacture, and a kit forpreventing and/or treating a fat metabolism disorder and its relatedconditions in a subject. Furthermore, the present invention also relatesto a plasminogen for preventing and/or treating a fat metabolismdisorder and its related conditions in a subject. The present inventionfurther relates to a medicament, a pharmaceutical composition, anarticle of manufacture, and a kit comprising plasminogen which areuseful for preventing and/or treating a fat metabolism disorder and itsrelated conditions in a subject.

In some embodiments, the fat metabolism disorder is a fat metabolismdisorder elicited or accompanied by an endocrine disorder disease, aglucose metabolism disease, a liver disease, a kidney disease, acardiovascular disease, an intestinal disease, a thyroid disease, agallbladder or a biliary tract disease, obesity, drinking, and a drugtherapy. In some embodiments, the fat metabolism disorder is a fatmetabolism disorder elicited or accompanied by hypertension, diabetesmellitus, chronic hepatitis, hepatic cirrhosis, renal injury, chronicglomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renalinsufficiency, kidney transplantation, uremia, hypothyroidism,obstructive cholecystitis, obstructive cholangitis, and a drug orhormone therapy. In some embodiments, the fat metabolism disorder ishyperlipemia, hyperlipoproteinemia, fatty liver, atherosclerosis,obesity, and a visceral fat deposition. In still some embodiments, theatherosclerosis comprises aortic atherosclerosis, coronaryatherosclerosis, cerebral atherosclerosis, renal atherosclerosis,hepatic atherosclerosis, mesenteric atherosclerosis, and lower limbatherosclerosis.

In yet another aspect, the present invention relates to a method forpreventing and/or reducing an abnormal fat deposition in a body tissueand an organ of a subject, comprising administering an effective amountof plasminogen to the subject. The present invention further relates tothe use of plasminogen for preventing and/or reducing an abnormal fatdeposition in a body tissue and an organ of a subject. The presentinvention further relates to the use of plasminogen in the preparationof a medicament, a pharmaceutical composition, an article ofmanufacture, and a kit for preventing and/or reducing an abnormal fatdeposition in a body tissue and an organ of a subject. Furthermore, thepresent invention also relates to a plasminogen for preventing and/orreducing an abnormal fat deposition in a body tissue and an organ of asubject. The present invention further relates to a medicament, apharmaceutical composition, an article of manufacture, and a kitcomprising plasminogen which are useful for preventing and/or reducingan abnormal fat deposition in a body tissue and an organ of a subject.

In yet another aspect, the present invention relates to a method forpreventing and/or treating a condition caused by an abnormal fatdeposition in a body tissue and an organ of a subject, comprisingadministering an effective amount of plasminogen to the subject. Thepresent invention further relates to the use of plasminogen forpreventing and/or treating a condition caused by an abnormal fatdeposition in a body tissue and an organ of a subject. The presentinvention further relates to the use of plasminogen in the preparationof a medicament, a pharmaceutical composition, an article ofmanufacture, and a kit for preventing and/or treating a condition causedby an abnormal fat deposition in a body tissue and an organ of asubject. Furthermore, the present invention also relates to amedicament, a pharmaceutical composition, an article of manufacture, anda kit comprising plasminogen which are useful for preventing and/ortreating a condition caused by an abnormal fat deposition in a bodytissue and an organ of a subject.

In some embodiments, the abnormal fat deposition in a body tissue and anorgan refers to an abnormal fat deposition in blood, a subcutaneoustissue, a vascular wall and an internal organ. In some embodiments, thecondition resulting from the abnormal fat deposition in a body tissueand an organ comprises obesity, hyperlipemia, hyperlipoproteinemia,fatty liver, atherosclerosis, a lipid-induced cardiac damage, alipid-induced renal damage, and a lipid-induced islet damage.

In yet another aspect, the present invention relates to a method forpreventing and/or treating a condition resulting from a fat metabolismdisorder in a subject, comprising administering an effective amount ofplasminogen to the subject. The present invention further relates to theuse of plasminogen for preventing and/or treating a condition resultingfrom a fat metabolism disorder in a subject. The present inventionfurther relates to the use of plasminogen in the preparation of amedicament, a pharmaceutical composition, an article of manufacture, anda kit for preventing and/or treating a condition resulting from a fatmetabolism disorder in a subject. Furthermore, the present inventionalso relates to a plasminogen for preventing and/or treating a conditionresulting from a fat metabolism disorder in a subject. The presentinvention further relates to a medicament, a pharmaceutical composition,an article of manufacture, and a kit comprising plasminogen which areuseful for preventing and/or treating a condition resulting from a fatmetabolism disorder in a subject. In some embodiments, the conditioncomprises obesity, hyperlipemia, hyperlipoproteinemia, fatty liver,atherosclerosis, a lipid-induced heart tissue injury, and alipid-induced renal injury.

In yet another aspect, the present invention relates to a method fortreating a disease in a subject by reducing an abnormal fat deposition,comprising administering an effective amount of plasminogen to thesubject. The present invention further relates to the use of plasminogenfor treating a disease in a subject by reducing an abnormal fatdeposition. The present invention further relates to the use ofplasminogen in the preparation of a medicament, a pharmaceuticalcomposition, an article of manufacture, and a kit for treating a diseasein a subject by reducing an abnormal fat deposition. Furthermore, thepresent invention also relates to a plasminogen for treating a diseasein a subject by reducing an abnormal fat deposition. The presentinvention further relates to a medicament, a pharmaceutical composition,an article of manufacture, and a kit comprising plasminogen which areuseful for treating a disease in a subject by reducing an abnormal fatdeposition.

In some embodiments, the disease comprises atherosclerosis, coronaryheart disease, angina pectoris, myocardial infarction, arrhythmia, fattyliver, hepatic cirrhosis, cerebral ischemia, cerebral infarction, renalinsufficiency, nephrotic syndrome, renal insufficiency, and obesity.

In yet another aspect, the present invention relates to a method forpreventing and/or treating a lipid-induced injury in a tissue and anorgan of a subject, comprising administering an effective amount ofplasminogen to the subject. The present invention further relates to theuse of plasminogen for preventing and/or treating a lipid-induced injuryin a tissue and an organ of a subject. The present invention furtherrelates to the use of plasminogen in the preparation of a medicament, apharmaceutical composition, an article of manufacture, and a kit forpreventing and/or treating a lipid-induced injury in a tissue and anorgan of a subject. Furthermore, the present invention also relates to aplasminogen for preventing and/or treating a lipid-induced injury in atissue and an organ of a subject. The present invention further relatesto a medicament, a pharmaceutical composition, an article ofmanufacture, and a kit comprising plasminogen which are useful forpreventing and/or treating a lipid-induced injury in a tissue and anorgan of a subject.

In some embodiments, the tissue and the organ comprise an arterial wall,a heart, a liver, a kidney, and a pancreas.

In yet another aspect, the present invention relates to a method forimproving hyperlipemia in a subject, comprising administering aneffective amount of plasminogen to the subject. The present inventionfurther relates to the use of plasminogen for improving hyperlipemia ina subject. The present invention further relates to the use ofplasminogen in the preparation of a medicament, a pharmaceuticalcomposition, an article of manufacture, and a kit for improvinghyperlipemia in a subject. Furthermore, the present invention alsorelates to a plasminogen for improving hyperlipemia in a subject. Thepresent invention further relates to a medicament, a pharmaceuticalcomposition, an article of manufacture, and a kit comprising plasminogenwhich are useful for improving hyperlipemia in a subject.

In some embodiments, the hyperlipemia is selected from one or more of:hypercholesterolemia, hypertriglyceridemia, combined hyperlipemia, andhypo-high-density lipoproteinemia.

In yet another aspect, the present invention relates to a method forreducing the risk of atherosclerosis in a subject, comprisingadministering an effective amount of plasminogen to the subject. Thepresent invention further relates to the use of plasminogen for reducingthe risk of atherosclerosis in a subject. The present invention furtherrelates to the use of plasminogen in the preparation of a medicament, apharmaceutical composition, an article of manufacture, and a kit forreducing the risk of atherosclerosis in a subject. Furthermore, thepresent invention also relates to a plasminogen for reducing the risk ofatherosclerosis in a subject. The present invention further relates to amedicament, a pharmaceutical composition, an article of manufacture, anda kit comprising plasminogen which are useful for reducing the risk ofatherosclerosis in a subject.

In some embodiments, the subject suffers from hypertension, obesity,diabetes mellitus, chronic hepatitis, hepatic cirrhosis, renal injury,chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome,renal insufficiency, kidney transplantation, uremia, hypothyroidism,obstructive cholecystitis, or obstructive cholangitis, or the subjecttakes a drug or hormone that affects fat metabolism. In someembodiments, the plasminogen reduces the risk of atherosclerosis in asubject in one or more ways selected from: lowering a total cholesterollevel, a triglyceride level, and a low-density lipoprotein level inblood, and elevating a high-density lipoprotein level in blood.

In yet another aspect, the present invention relates to a method fortreating a disease in a subject by improving hyperlipemia, comprisingadministering an effective amount of plasminogen to the subject. Thepresent invention further relates to the use of plasminogen for treatinga disease by improving hyperlipemia in a subject. The present inventionfurther relates to the use of plasminogen in the preparation of amedicament, a pharmaceutical composition, an article of manufacture, anda kit for treating a disease by improving hyperlipemia in a subject.Furthermore, the present invention also relates to a plasminogen fortreating a disease by improving hyperlipemia in a subject. The presentinvention further relates to a medicament, a pharmaceutical composition,an article of manufacture, and a kit comprising plasminogen which areuseful for treating a disease by improving hyperlipemia in a subject.

In some embodiments, the condition comprises diabetes mellitus,hypertension, atherosclerosis, coronary heart disease, angina pectoris,myocardial infarction, arrhythmia, chronic hepatitis, fatty liver,hepatic cirrhosis, cerebral circulation insufficiency, cerebralischemia, cerebral infarction, chronic nephritis, chronicpyelonephritis, renal insufficiency, nephrotic syndrome, uremia, andobesity.

In yet another aspect, the present invention relates to a method forpreventing and/or treating a hyperlipemia-related condition in asubject, comprising administering an effective amount of plasminogen tothe subject. The present invention further relates to the use ofplasminogen for preventing and/or treating a hyperlipemia-relatedcondition in a subject. The present invention further relates to the useof plasminogen in the preparation of a medicament, a pharmaceuticalcomposition, an article of manufacture, and a kit for preventing and/ortreating a hyperlipemia-related condition in a subject. Furthermore, thepresent invention also relates to a plasminogen for preventing and/ortreating a hyperlipemia-related condition in a subject. The presentinvention further relates to a medicament, a pharmaceutical composition,an article of manufacture, and a kit comprising plasminogen which areuseful for preventing and/or treating a hyperlipemia-related conditionin a subject. In some embodiments, the condition comprises diabetesmellitus, hypertension, atherosclerosis, coronary heart disease, anginapectoris, myocardial infarction, arrhythmia, chronic hepatitis, fattyliver, hepatic cirrhosis, cerebral circulation insufficiency, cerebralischemia, cerebral infarction, chronic nephritis, chronicpyelonephritis, renal insufficiency, nephrotic syndrome, uremia, andobesity.

In any of the above-mentioned embodiments of the present invention, theplasminogen is administered in combination with one or more other drugsor therapies. In some embodiments, the one or more other drugs comprisesa drug for treating hypertension, a drug for treating diabetes mellitus,a drug for treating atherosclerosis, a drug for treating chronicglomerulonephritis, a drug for treating chronic pyelonephritis, a drugfor treating nephrotic syndrome, a drug for treating renalinsufficiency, a drug for treating uremia, a drug for treating kidneytransplantation, a drug for treating fatty liver, a drug for treatinghepatic cirrhosis, and a drug for treating obesity. In some embodiments,the other drugs comprise: a hypolipidemic drug, an anti-platelet drug,an antihypertensive drug, a vasodilator, a hypoglycemic drug, ananticoagulant drug, a thrombolytic drug, a hepatoprotective drug, ananti-arrhythmia drug, a cardiotonic drug, a diuretic drug, ananti-infective drug, an antiviral drug, an immunomodulatory drug, aninflammatory regulatory drug, an anti-tumor drug, a hormone drug, andthyroxine. In some further embodiments, the drugs comprise hypolipidemicdrugs: statins; fibrates; niacin; cholestyramine; clofibrate;unsaturated fatty acids such as Yishouning, Xuezhiping, and Xinmaile;and alginic sodium diester; anti-platelet drugs: aspirin; dipyridamole;clopidogrel; and cilostazol; vasodilators: hydralazine; nitroglycerin,and isosorbide dinitrate; sodium nitroprusside; α1-receptor blockerssuch as prazosin; α-receptor blockers such as phentolamine; β2-receptorstimulants such as salbutamol; captopril, enalapril; nifedipine,diltiazem; and salbutamol, loniten, prostaglandin, and atrialnatriuretic peptide; thrombolytic drugs: urokinase, and streptokinase;tissue-type plasminogen activators; single chain urokinase-typeplasminogen activators; and a TNK tissue-type plasminogen activator; andanticoagulant drugs: heparin; enoxaparin; nadroparin; and bivalirudin.

In any of the above-mentioned embodiments of the present invention, theplasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% sequence identity with SEQ ID No. 2, 6, 8, 10 or 12, and still havethe activity of plasminogen. In some embodiments, the plasminogen is aprotein that has 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35,1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid added,deleted and/or substituted in SEQ ID No. 2, 6, 8, 10 or 12, and stillhas the activity of plasminogen.

In some embodiments, the plasminogen is a protein that comprises aplasminogen active fragment and still has the activity of plasminogen.In some embodiments, the plasminogen is selected from Glu-plasminogen,Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogenor their variants that retain the plasminogen activity. In someembodiments, the plasminogen is a natural or synthetic humanplasminogen, or a variant or fragment thereof that still retains theplasminogen activity. In some embodiments, the plasminogen is anortholog of human plasminogen from a primate or a rodent, or a variantor fragment thereof that still retains the plasminogen activity. In someembodiments, the amino acids of the plasminogen are as shown in SEQ IDNo. 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is a naturalhuman plasminogen.

In some embodiments, the subject is a human. In some embodiments, thesubject is lack of or deficient in plasminogen. In some embodiments, thelack or deficiency is congenital, secondary and/or local.

In some embodiments, the pharmaceutical composition comprises apharmaceutically acceptable carrier and the plasminogen for use in theabove-mentioned method. In some embodiments, the kit may be a preventiveor therapeutic kit comprising: (i) the plasminogen for use in theabove-mentioned method, and (ii) a means for delivering the plasminogento the subject. In some embodiments, the means is a syringe or a vial.In some embodiments, the kit further comprises a label or an instructionfor use indicating the administration of the plasminogen to the subjectto implement any one of the above-mentioned methods.

In some embodiments, the article of manufacture comprising: a containercomprising a label; and (i) the plasminogen for use in theabove-mentioned methods or a pharmaceutical composition comprising theplasminogen, wherein the label indicates the administration of theplasminogen or the composition to the subject to implement any one ofthe above-mentioned methods.

In some embodiments, the kit or the article of manufacture furthercomprises one or more additional means or containers containing otherdrugs. In some embodiments, the other drugs are selected from a groupof: a hypolipidemic drug, an anti-platelet drug, an antihypertensivedrug, a vasodilator, a hypoglycemic drug, an anticoagulant drug, athrombolytic drug, a hepatoprotective drug, an anti-arrhythmia drug, acardiotonic drug, a diuretic drug, an anti-infective drug, an antiviraldrug, an immunomodulatory drug, an inflammatory regulatory drug, ananti-tumor drug, a hormone drug, and thyroxine.

In some embodiments of the above-mentioned method, the plasminogen isadministered by systemic or topical route, preferably by the followingroutes: intravenous, intramuscular, and subcutaneous administration ofplasminogen for treatment. In some embodiments of the above-mentionedmethod, the plasminogen is administered in combination with a suitablepolypeptide carrier or stabilizer. In some embodiments of theabove-mentioned method, the plasminogen is administered at a dosage of0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200mg/kg, 1-100 mg/kg or 10-100 mg/kg (by per kg of body weight) or0.0001-2000 mg/cm², 0.001-800 mg/cm², 0.01-600 mg/cm², 0.1-400 mg/cm²,1-200 mg/cm², 1-100 mg/cm² or 10-100 mg/cm² (by per square centimeter ofbody surface area) daily, preferably the dosage is repeated at leastonce, preferably the dosage is administered at least daily.

The present invention explicitly encompasses all the combinations oftechnical features belonging to the embodiments of the presentinvention, and these combined technical solutions have been explicitlydisclosed in the present application, as if the above-mentionedtechnical solutions were individually and explicitly disclosed. Inaddition, the present invention also explicitly encompasses all thecombinations between various embodiments and elements thereof, and thecombined technical solutions are explicitly disclosed herein.

Definition

The “fat metabolism disorder” of the present invention, also known as“abnormal fat metabolism” and “lipodystrophy”, is the generic term forthe clinical or pathological manifestations caused by the abnormality,disorder or dysfunction of fat metabolism. “Fat metabolism disorder”,“abnormal fat metabolism”, and “lipodystrophy” are used interchangeablyherein. “Fat metabolism”, “lipid metabolism”, and “metabolism of lipids”are used interchangeably in the present invention.

“A fat metabolism disorder-related condition” is the generic term forthe conditions related to fat metabolism disorder. The expression“related” may be etiology-, pathogenesis-, pathogenic manifestation-,clinical symptom- and/or therapeutic principle-related.

“Blood lipid” is the generic term for triglycerides, cholesterol andphospholipids. Lipoprotein is a globular macromolecular complex composedof apolipoproteins and blood lipids. Since lipoprotein is composed ofdifferent components, cholesterol and triglycerides, at differentdensities, it is divided into categories: chylomicron (CM), verylow-density lipoprotein (VLDL), intermediate density lipoprotein (IDL),low-density lipoprotein (LDL), and high-density lipoprotein (HDL).According to the blood lipid risk level, the most common clinical typesof dyslipoproteinemia are: hypercholesterolemia, hypertriglyceridemia,combined hyperlipemia, and hypo-high-density lipoproteinemia. Secondarydyslipidemia is commonly found in diabetes mellitus, hypothyroidism,nephrotic syndrome, kidney transplantation, a severe liver disease, anobstructive biliary tract disease, obesity, drinking, and drug therapysuch as oestrogen therapy, etc. Primary dyslipidemia can be consideredif secondary dyslipidemia can be ruled out.

“Hyperlipemia” refers to a pathological condition in which blood lipidcomponents such as cholesterol, triglycerides, phospholipids andnon-lipidated fatty acids are elevated in plasma.

“A hyperlipemia-related condition” refers to a condition of whichetiology, pathogenesis, pathogenic manifestations, clinical symptomsand/or therapeutic principle are related to hyperlipemia. Preferably,the condition includes but is not limited to diabetes mellitus,hypertension, atherosclerosis, coronary heart disease, angina pectoris,myocardial infarction, arrhythmia, chronic hepatitis, fatty liver,hepatic cirrhosis, cerebral circulation insufficiency, cerebralischemia, cerebral infarction, chronic nephritis, chronicpyelonephritis, renal insufficiency, nephrotic syndrome, uremia, andobesity.

Abnormalities of one or several lipids in plasma due to abnormal fatmetabolism or turnover are referred to as “hyperlipemia”,“hyperlipidemia” or “dyslipidemia”.

Lipids are insoluble or slightly soluble in water, and must bind toproteins to form lipoproteins to function in the blood circulation.Therefore, hyperlipemia is often a reflection of “hyperlipoproteinemia”.

The “hyperlipemia-related condition” of the present invention is alsoknown as “hyperlipidemia-related condition” and“hyperlipoproteinemia-related condition”.

“Fatty liver” refers to a lesion of excessive accumulation of fat inhepatocytes due to various causes. It can be an independent disease orcan be caused by other causes, such as obesity-induced fatty liver,alcohol-induced fatty liver, rapid weight loss induced fatty liver,malnutrition-induced fatty liver, diabetic fatty liver, drug-inducedfatty liver, etc.

In the case of fatty liver, the lipid droplets in the hepatocytes areincreased, resulting in steatosis and enlargement of the hepatocytes,and extrusion of the nuclei away from the center. Fat metabolism mainlytakes place in the mitochondria. Fat is transported out of the cellmainly through the smooth endoplasmic reticulum. Fat accumulation inhepatocytes further aggravates the burden of mitochondria andendoplasmic reticulum and reduces their functions, thus affecting themetabolism of other nutrients, hormones and vitamins. Long-termhepatocyte degeneration will lead to regeneration disorder and necrosisof hepatocytes, and thus form liver fibrosis and hepatic cirrhosis.

“Atherosclerosis” is a chronic, progressive arterial disease in whichthe fat deposited in the arteries partially or completely blocks bloodflow. Atherosclerosis occurs when the otherwise smooth and solidarterial intima becomes roughened and thickened and is blocked by fat,fibrin, calcium, and cellular debris. Atherosclerosis is a progressiveprocess. When the concentration of lipids in the blood is greatlyincreased, fatty streaks form along the arterial wall. These streaks canlead to deposits of fat and cholesterol, which attach to the otherwisesmooth arterial intima and thus form nodules. Underneath these nodules,fibrotic scar tissue develops, leading to calcium deposition. Thecalcium deposits gradually develop into a chalky hard film (referred toas atherosclerotic plaque) that cannot be removed. This permanent filminside the artery would block the normal expansion and contraction ofthe artery, which slows the blood flow velocity within the artery,making the blood easy to form clots that block or stop blood flowingthrough the artery.

The exact cause of atherosclerosis has not been determined. However,important pathogenic factors have been identified as hyperlipemia,hypertension, a history of smoking, a family history of atherosclerosis(suffering from the disease before the age of 60) or diabetes mellitus.Hyperlipemia can promote the formation of fatty streaks. Hypertensionexerts a constant force on the arteries, accelerating the process ofarterial occlusion and arteriosclerosis; therefore, it can increase theprevalence of atherosclerosis. Smoking can cause arterial contractionsand restrict blood flow, thus setting the stage for arterial occlusion.Diabetes mellitus can also contribute to the development ofatherosclerosis, especially in very small arteries.

In the case of atherosclerosis alone, people do not feel any symptoms.The disease is only discovered when an artery connected to a vital organin the body is blocked. Symptoms are more pronounced when arteries inthe organ are blocked. For instance, people may feel angina pectoris ifthe cardiac feeding artery is partially blocked; however, if it iscompletely blocked, it may lead to a heart disease (the death of hearttissue fed by the blocked artery). If atherosclerosis affects thecerebral arteries, people may experience dizziness, blurred vision,syncope, and even a stroke (the death of brain tissue fed by the blockedarteries, resulting in a nerve damage, such as paralysis of a limbcontrolled by dead brain tissue). Occlusion of arteries to the kidneysmay also lead to renal failure. Occlusion of blood vessels to the eyesmay lead to blindness. Occlusion of arteries in the extremities may leadto lesions in each limb.

Atherosclerosis is the main cause of coronary heart disease, cerebralinfarction, and peripheral vascular disease. Lipid metabolism disorderis the pathological basis of atherosclerosis, wherein the lesion ofaffected artery begins from intima, where accumulation of lipids andcompound carbohydrates, hemorrhage and thrombosis first appeargenerally, followed by hyperplasia of fibrous tissue and calcinosis,with gradual metamorphosis and calcification of the arterial mediallayer, leading to thickening and hardening of the arterial wall, andstenosis of vascular lumen. The lesion generally involves the large andmedium muscular arteries. Once the lesion has developed enough to blockthe arterial lumen, the tissues or organs supplied by the artery willbecome ischemic or necrotic.

Atherosclerosis is a systemic disease, and the occurrence of anatherosclerotic lesion in the blood vessels of an organ means that bloodvessels elsewhere may already have had the same lesion; similarly, avascular event in an organ means an increased risk of vascular eventelsewhere.

DETAILED DESCRIPTION OF EMBODIMENTS

Plasmin is a key component of the plasminogen activation system (PAsystem). It is a broad-spectrum protease that can hydrolyze severalcomponents of the extracellular matrix (ECM), including fibrin, gelatin,fibronectin, laminin, and proteoglycan^([9]). In addition, plasmin canactivate some pro-matrix metalloproteinases (pro-MMPs) to form activematrix metalloproteinases (MMPs). Therefore, plasmin is considered to bean important upstream regulator of extracellular proteolysis^([10,11]).Plasmin is formed by the proteolysis of plasminogen by two physiologicalPAs: tissue plasminogen activator (tPA) or urokinase-type plasminogenactivator (uPA). Due to the relatively high level of plasminogen inplasma and other body fluids, it is traditionally believed that theregulation of the PA system is primarily achieved through the levels ofPA synthesis and activity. The synthesis of PA system components isstrictly regulated by different factors, such as hormones, growthfactors and cytokines. In addition, there are also specificphysiological inhibitors of plasmin and PAs. The main inhibitor ofplasmin is α2-antiplasmin. The activity of PAs is simultaneouslyinhibited by the plasminogen activator inhibitor-1 (PAI-1) of uPA andtPA and regulated by the plasminogen activator inhibitor-2 (PAI-2) thatprimarily inhibits uPA. There are uPA-specific cell surface receptors(uPARs) that have direct hydrolytic activity on certain cellsurfaces^([12,13]).

Plasminogen is a single-stranded glycoprotein composed of 791 aminoacids and has a molecular weight of about 92 kDa^([14,15]). Plasminogenis mainly synthesized in the liver and is abundantly present in theextracellular fluid. The content of plasminogen in plasma is about 2 μM.Therefore, plasminogen is a huge potential source of proteolyticactivity in tissues and body fluids^([16,17]). Plasminogen exists in twomolecular forms: glutamic acid-plasminogen (Glu-plasminogen) andlysine-plasminogen (Lys-plasminogen). The naturally secreted anduncleaved forms of plasminogen have an amino-terminal (N-terminal)glutamic acid and are therefore referred to as glutamicacid-plasminogen. However, in the presence of plasmin, glutamicacid-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77.Compared with glutamic acid-plasminogen, lysine-plasminogen has a higheraffinity for fibrin and can be activated by PAs at a higher rate. TheArg560-Val561 peptide bond between these two forms of plasminogen can becleaved by uPA or tPA, resulting in the formation of plasmin as adisulfide-linked double-strand protease^([18]). The amino-terminalportion of plasminogen contains five homotrimeric rings, i.e., theso-called kringles, and the carboxy-terminal portion contains a proteasedomain. Some kringles contain lysine-binding sites that mediate thespecific interaction of plasminogen with fibrin and its inhibitor α2-AP.A newly discovered plasminogen is a 38 kDa fragment, comprising kringles1-4, is a potent inhibitor of angiogenesis. This fragment is named asangiostatin and can be produced by proteolysis of plasminogen by severalproteases.

The main substrate of plasmin is fibrin, and the dissolution of fibrinis the key to prevent pathological thrombosis^([19]). Plasmin also hassubstrate specificity for several components of ECM, including laminin,fibronectin, proteoglycan and gelatin, indicating that plasmin alsoplays an important role in ECM remodeling^([15,20,21]). Indirectly,plasmin can also degrade other components of ECM by converting certainprotease precursors into active proteases, including MMP-1, MMP-2, MMP-3and MMP-9. Therefore, it has been proposed that plasmin may be animportant upstream regulator of extracellular proteolysis^([22]). Inaddition, plasmin has the ability to activate certain potential forms ofgrowth factors^([23-25]). In vitro, plasmin can also hydrolyzecomponents of the complement system and release chemotactic complementfragments.

“Plasmin” is a very important enzyme that exists in the blood and canhydrolyze fibrin clots into fibrin degradation products and D-dimers.

“Plasminogen” is the zymogenic form of plasmin, and based on thesequence in the swiss prot and calculated from the amino acid sequence(SEQ ID No. 4) of the natural human plasminogen containing a signalpeptide, is a glycoprotein composed of 810 amino acids, which has amolecular weight of about 90 kD and is synthesized mainly in the liverand capable of circulating in the blood; and the cDNA sequence encodingthis amino acid sequence is as shown in SEQ ID No. 3. Full-lengthplasminogen contains seven domains: a C-terminal serine protease domain,an N-terminal Pan Apple (PAp) domain and five Kringle domains (Kringles1-5). Referring to the sequence in the swiss prot, the signal peptidecomprises residues Met1-Gly19, PAp comprises residues Glu20-Val98,Kringle 1 comprises residues Cys103-Cys181, Kringle 2 comprises residuesGlu184-Cys262, Kringle 3 comprises residues Cys275-Cys352, Kringle 4comprises residues Cys377-Cys454, and Kringle 5 comprises residuesCys481-Cys560. According to the NCBI data, the serine protease domaincomprises residues Val581-Arg804.

Glu-plasminogen is a natural full-length plasminogen and is composed of791 amino acids (without a signal peptide of 19 amino acids); the cDNAsequence encoding this sequence is as shown in SEQ ID No. 1; and theamino acid sequence is as shown in SEQ ID No. 2. In vivo,Lys-plasminogen, which is formed by hydrolysis of amino acids atpositions 76-77 of Glu-plasminogen, is also present, as shown in SEQ IDNo.6; and the cDNA sequence encoding this amino acid sequence is asshown in SEQ ID No.5. δ-plasminogen is a fragment of full-lengthplasminogen that lacks the structure of Kringle 2-Kringle 5 and containsonly Kringle 1 and the serine protease domain^([26,27]). The amino acidsequence (SEQ ID No. 8) of 6-plasminogen has been reported in theliterature^([27]), and the cDNA sequence encoding this amino acidsequence is as shown in SEQ ID No. 7. Mini-plasminogen is composed ofKringle 5 and the serine protease domain, and has been reported in theliterature to comprise residues Val443-Asn791 (with the Glu residue ofthe Glu-plasminogen sequence that does not contain a signal peptide asthe starting amino acid)^([28]); the amino acid sequence is as shown inSEQ ID No. 10; and the cDNA sequence encoding this amino acid sequenceis as shown in SEQ ID No. 9. Micro-plasminogen comprises only the serineprotease domain, the amino acid sequence of which has been reported inthe literature to comprise residues Ala543-Asn791 (with the Glu residueof the Glu-plasminogen sequence that does not contain a signal peptideas the starting amino acid)^([29]), and the sequence of which has beenalso reported in patent document CN 102154253 A to comprise residuesLys531-Asn791 (with the Glu residue of the Glu-plasminogen sequence thatdoes not contain a signal peptide as the starting amino acid) (thesequence in this patent application refers to the patent document CN102154253 A); the amino acid sequence is as shown in SEQ ID No. 12; andthe cDNA sequence encoding this amino acid sequence is as shown in SEQID No. 11.

In the present invention, “plasmin” is used interchangeably with“fibrinolysin” and “fibrinoclase”, and the terms have the same meaning;and “plasminogen” is used interchangeably with “plasminogen” and“fibrinoclase zymogen”, and the terms have the same meaning.

In the present application, the meaning of “lack” in plasminogen is thatthe content or activity of plasminogen in the body of a subject is lowerthan that of a normal person, which is low enough to affect the normalphysiological function of the subject; and the meaning of “deficiency”in plasminogen is that the content or activity of plasminogen in thebody of a subject is significantly lower than that of a normal person,or even the activity or expression is extremely small, and only throughexogenous supply can the normal physiological function be maintained.

Those skilled in the art can understand that all the technical solutionsof the plasminogen of the present invention are suitable for plasmin.Therefore, the technical solutions described in the present inventioncover plasminogen and plasmin.

In the course of circulation, plasminogen is in a closed, inactiveconformation, but when bound to thrombi or cell surfaces, it isconverted into an active plasmin in an open conformation under themediation of a plasminogen activator (PA). The active plasmin canfurther hydrolyze the fibrin clots to fibrin degradation products andD-dimers, thereby dissolving the thrombi. The PAp domain of plasminogencomprises an important determinant that maintains plasminogen in aninactive, closed conformation, and the KR domain is capable of bindingto lysine residues present on receptors and substrates. A variety ofenzymes that can serve as plasminogen activators are known, including:tissue plasminogen activator (tPA), urokinase plasminogen activator(uPA), kallikrein, coagulation factor XII (Hagmann factor), and thelike.

“Plasminogen active fragment” refers to an active fragment in theplasminogen protein that is capable of binding to a target sequence in asubstrate and exerting the proteolytic function. The technical solutionsof the present invention involving plasminogen encompass technicalsolutions in which plasminogen is replaced with a plasminogen activefragment. The plasminogen active fragment of the present invention is aprotein comprising a serine protease domain of plasminogen. Preferably,the plasminogen active fragment of the present invention comprises SEQID No.14, or an amino acid sequence having an amino acid sequenceidentity of at least 80%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ IDNo.14. Therefore, plasminogen of the present invention comprises aprotein containing the plasminogen active fragment and still having theplasminogen activity.

At present, methods for determining plasminogen and its activity inblood include: detection of tissue plasminogen activator activity(t-PAA), detection of tissue plasminogen activator antigen (t-PAAg) inplasma, detection of tissue plasminogen activity (plgA) in plasma,detection of tissue plasminogen antigen (plgAg) in plasma, detection ofactivity of the inhibitor of tissue plasminogen activators in plasma,detection of inhibitor antigens of tissue plasminogen activators inplasma and detection of plasmin-anti-plasmin (PAP) complex in plasma.The most commonly used detection method is the chromogenic substratemethod: streptokinase (SK) and a chromogenic substrate are added to atest plasma, the PLG in the test plasma is converted into PLM by theaction of SK, PLM acts on the chromogenic substrate, and then it isdetermined that the increase in absorbance is directly proportional toplasminogen activity using a spectrophotometer. In addition, plasminogenactivity in blood can also be determined by immunochemistry, gelelectrophoresis, immunonephelometry, radioimmuno-diffusion and the like.

“Orthologues or orthologs” refer to homologs between different species,including both protein homologs and DNA homologs, and are also known asorthologous homologs and vertical homologs. The term specifically refersto proteins or genes that have evolved from the same ancestral gene indifferent species. The plasminogen of the present invention includeshuman natural plasminogen, and also includes orthologues or orthologs ofplasminogens derived from different species and having plasminogenactivity.

“Conservatively substituted variant” refers to one in which a givenamino acid residue is changed without altering the overall conformationand function of the protein or enzyme, including, but not limited to,replacing an amino acid in the amino acid sequence of the parent proteinby an amino acid with similar properties (such as acidity, alkalinity,hydrophobicity, etc.). Amino acids with similar properties are wellknown. For example, arginine, histidine and lysine are hydrophilic basicamino acids and are interchangeable. Similarly, isoleucine is ahydrophobic amino acid that can be replaced by leucine, methionine orvaline. Therefore, the similarity of two proteins or amino acidsequences with similar functions may be different. For example, thesimilarity (identity) is 70%-99% based on the MEGALIGN algorithm.“Conservatively substituted variant” also includes a polypeptide orenzyme having amino acid identity of 60% or more, preferably 75% ormore, more preferably 85% or more, even more preferably 90% or more asdetermined by the BLAST or FASTA algorithm, and having the same orsubstantially similar properties or functions as the natural or parentprotein or enzyme.

“Isolated” plasminogen refers to the plasminogen protein that isisolated and/or recovered from its natural environment. In someembodiments, the plasminogen will be purified (1) to a purity of greaterthan 90%, greater than 95% or greater than 98% (by weight), asdetermined by the Lowly method, such as more than 99% (by weight); (2)to a degree sufficiently to obtain at least 15 residues of theN-terminal or internal amino acid sequence using a spinning cupsequenator; or (3) to homogeneity, which is determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing ornon-reducing conditions using Coomassie blue or silver staining Isolatedplasminogen also includes plasminogen prepared from recombinant cells bybioengineering techniques and separated by at least one purificationstep.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein and refer to polymeric forms of amino acids ofany length, which may include genetically encoded and non-geneticallyencoded amino acids, chemically or biochemically modified or derivatizedamino acids, and polypeptides having modified peptide backbones. Theterm includes fusion proteins, including, but not limited to, fusionproteins having heterologous amino acid sequences, fusions havingheterologous and homologous leader sequences (with or without N-terminalmethionine residues); and the like.

The “percent amino acid sequence identity (%)” with respect to thereference polypeptide sequence is defined as the percentage of aminoacid residues in the candidate sequence identical to the amino acidresidues in the reference polypeptide sequence when a gap is introducedas necessary to achieve maximal percent sequence identity and noconservative substitutions are considered as part of sequence identity.The comparison for purposes of determining percent amino acid sequenceidentity can be achieved in a variety of ways within the skill in theart, for example using publicly available computer softwares, such asBLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for aligning sequences,including any algorithm needed to achieve the maximum comparison overthe full length of the sequences being compared. However, for purposesof the present invention, the percent amino acid sequence identity valueis generated using the sequence comparison computer program ALIGN-2.

In the case of comparing amino acid sequences using ALIGN-2, the % aminoacid sequence identity of a given amino acid sequence A relative to agiven amino acid sequence B (or may be expressed as a given amino acidsequence A having or containing a certain % amino acid sequence identityrelative to, with or for a given amino acid sequence B) is calculated asfollows:

fraction X/Y×100

wherein X is the number of identically matched amino acid residuesscored by the sequence alignment program ALIGN-2 in the alignment of Aand B using the program, and wherein Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A relative to B will not be equal tothe % amino acid sequence identity of B relative to A. Unlessspecifically stated otherwise, all the % amino acid sequence identityvalues used herein are obtained using the ALIGN-2 computer program asdescribed in the previous paragraph.

As used herein, the terms “treatment” and “treating” refer to obtaininga desired pharmacological and/or physiologic effect. The effect may becomplete or partial prevention of a disease or its symptoms and/orpartial or complete cure of the disease and/or its symptoms, andincludes: (a) prevention of the disease from developing in a subjectthat may have a predisposition to the disease but has not been diagnosedas having the disease; (b) suppression of the disease, i.e., blockingits formation; and (c) alleviation of the disease and/or its symptoms,i.e., eliminating the disease and/or its symptoms.

The terms “individual”, “subject” and “patient” are used interchangeablyherein and refer to mammals, including, but not limited to, murine (ratsand mice), non-human primates, humans, dogs, cats, hoofed animals (e.g.,horses, cattle, sheep, pigs, goats) and so on.

“Therapeutically effective amount” or “effective amount” refers to anamount of plasminogen sufficient to achieve the prevention and/ortreatment of a disease when administered to a mammal or another subjectto treat the disease. The “therapeutically effective amount” will varydepending on the plasminogen used, the severity of the disease and/orits symptoms, as well as the age, body weight of the subject to betreated, and the like.

Preparation of the Plasminogen of the Present Invention

Plasminogen can be isolated and purified from nature for furthertherapeutic uses, and can also be synthesized by standard chemicalpeptide synthesis techniques. When chemically synthesized, a polypeptidecan be subjected to liquid or solid phase synthesis. Solid phasepolypeptide synthesis (SPPS) is a method suitable for chemical synthesisof plasminogen, in which the C-terminal amino acid of a sequence isattached to an insoluble support, followed by the sequential addition ofthe remaining amino acids in the sequence. Various forms of SPPS, suchas Fmoc and Boc, can be used to synthesize plasminogen. Techniques forsolid phase synthesis are described in Barany and Solid-Phase PeptideSynthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol.2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J.Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al. Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and GanesanA. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero J A et al. 2005 ProteinPept Lett. 12:723-8. Briefly, small insoluble porous beads are treatedwith a functional unit on which a peptide chain is constructed. Afterrepeated cycles of coupling/deprotection, the attached solid phase freeN-terminal amine is coupled to a single N-protected amino acid unit.This unit is then deprotected to expose a new N-terminal amine that canbe attached to another amino acid. The peptide remains immobilized onthe solid phase before it is cut off.

Standard recombinant methods can be used to produce the plasminogen ofthe present invention. For example, a nucleic acid encoding plasminogenis inserted into an expression vector, so that it is operably linked toa regulatory sequence in the expression vector. Expression regulatorysequence includes, but is not limited to, promoters (e.g., naturallyassociated or heterologous promoters), signal sequences, enhancerelements and transcription termination sequences. Expression regulationcan be a eukaryotic promoter system in a vector that is capable oftransforming or transfecting eukaryotic host cells (e.g., COS or CHOcells). Once the vector is incorporated into a suitable host, the hostis maintained under conditions suitable for high-level expression of thenucleotide sequence and collection and purification of plasminogen.

A suitable expression vector is usually replicated in a host organism asan episome or as an integral part of the host chromosomal DNA. Ingeneral, an expression vector contains a selective marker (e.g.,ampicillin resistance, hygromycin resistance, tetracycline resistance,kanamycin resistance or neomycin resistance) to facilitate detection ofthose exogenous cells transformed with a desired DNA sequence.

Escherichia coli is an example of prokaryotic host cells that can beused to clone a polynucleotide encoding the subject antibody. Othermicrobial hosts suitable for use include Bacillus, for example, Bacillussubtilis and other species of enterobacteriaceae (such as Salmonellaspp. and Serratia spp.), and various Pseudomonas spp. In theseprokaryotic hosts, expression vectors can also be generated which willtypically contain an expression control sequence (e.g., origin ofreplication) that is compatible with the host cell. In addition, therewill be many well-known promoters, such as the lactose promoter system,the tryptophan (trp) promoter system, the beta-lactamase promoter systemor the promoter system from phage lambda. Optionally in the case ofmanipulation of a gene sequence, a promoter will usually controlexpression, and has a ribosome binding site sequence and the like toinitiate and complete transcription and translation.

Other microorganisms, such as yeast, can also be used for expression.Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitableyeast host cells, in which a suitable vector has an expression controlsequence (e.g., promoter), an origin of replication, a terminationsequence and the like, as required. A typical promoter comprises3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters specifically include promoters derived from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

In addition to microorganisms, mammalian cells (e.g., mammalian cellscultured in cell culture in vitro) can also be used to express andgenerate the anti-Tau antibody of the present invention (e.g., apolynucleotide encoding a subject anti-Tau antibody). See Winnacker,From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitablemammalian host cells include CHO cell lines, various Cos cell lines,HeLa cells, myeloma cell lines and transformed B cells or hybridomas.Expression vectors for these cells may comprise an expression controlsequence, such as an origin of replication, promoter and enhancer (Queenet al. Immunol. Rev. 89:49 (1986)), as well as necessary processinginformation sites, such as a ribosome binding site, RNA splice site,polyadenylation site and transcription terminator sequence. Examples ofsuitable expression control sequences are promoters derived from whiteimmunoglobulin gene, SV40, adenovirus, bovine papilloma virus,cytomegalovirus and the like. See Co et al. J. Immunol. 148:1149 (1992).

Once synthesized (chemically or recombinantly), the plasminogen of thepresent invention can be purified according to standard procedures inthe art, including ammonium sulfate precipitation, affinity column,column chromatography, high performance liquid chromatography (HPLC),gel electrophoresis and the like. The plasminogen is substantially pure,e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, atleast about 90% to 95% pure, or 98% to 99% pure or purer, for examplefree of contaminants such as cell debris, macromolecules other than thesubject antibody and the like.

Pharmaceutical Formulations

A therapeutic formulation can be prepared by mixing plasminogen of adesired purity with an optional pharmaceutical carrier, excipient orstabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.ed. (1980)) to form a lyophilized preparation or an aqueous solution.Acceptable carriers, excipients and stabilizers are non-toxic to therecipient at the dosages and concentrations employed, and includebuffers, such as phosphates, citrates and other organic acids;antioxidants, including ascorbic acid and methionine; preservatives(e.g., octadecyl dimethyl benzyl ammonium chloride; hexane chloridediamine; benzalkonium chloride and benzethonium chloride; phenol,butanol or benzyl alcohol; alkyl p-hydroxybenzoates, such as methyl orpropyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight polypeptides (less thanabout 10 residues); proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagine, histidine, arginineor lysine; monosaccharides, disaccharides and other carbohydrates,including glucose, mannose or dextrins; chelating agents, such as EDTA;sugars, such as sucrose, mannitol, fucose or sorbitol; salt-formingcounterions, such as sodium; metal complexes (e.g., zinc-proteincomplexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibodyformulations are described in WO 97/04801, which is incorporated hereinby reference.

The formulations of the invention may also comprise one or more activecompounds required for the particular condition to be treated,preferably those that are complementary in activity and have no sideeffects with one another, for example anti-hypertensive drugs,anti-arrhythmic drugs, drugs for treating diabetes mellitus, and thelike.

The plasminogen of the present invention may be encapsulated inmicrocapsules prepared by techniques such as coacervation or interfacialpolymerization, for example, it may be incorporated in a colloid drugdelivery system (e.g., liposomes, albumin microspheres, microemulsions,nanoparticles and nanocapsules), or incorporated inhydroxymethylcellulose or gel-microcapsules and poly-(methylmethacrylate) microcapsules in macroemulsions. These techniques aredisclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.Ed. (1980).

The plasminogen of the present invention for in vivo administration mustbe sterile. This can be easily achieved by filtration through a sterilefiltration membrane before or after freeze drying and reconstitution.

The plasminogen of the present invention can be prepared into asustained-release preparation. Suitable examples of sustained-releasepreparations include solid hydrophobic polymer semi-permeable matriceshaving a shape and containing glycoproteins, such as films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate)) (Langeret al. J. Biomed. Mater. Res., 15: 167-277 (1981); and Langer, Chem.Tech., 12:98-105 (1982)), or poly(vinyl alcohol), polylactides (U.S.Pat. No. 3,773,919, and EP 58,481), copolymer of L-glutamic acid and yethyl-L-glutamic acid (Sidman et al. Biopolymers 22:547(1983)),nondegradable ethylene-vinyl acetate (Langer et al. supra), ordegradable lactic acid-glycolic acid copolymers such as Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly D-(−)-3-hydroxybutyric acid. Polymers,such as ethylene-vinyl acetate and lactic acid-glycolic acid, are ableto persistently release molecules for 100 days or longer, while somehydrogels release proteins for a shorter period of time. A rationalstrategy for protein stabilization can be designed based on relevantmechanisms. For example, if the aggregation mechanism is discovered tobe formation of an intermolecular S—S bond through thio-disulfideinterchange, stability is achieved by modifying sulthydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

Administration and Dosage

The pharmaceutical composition of the present invention is administeredin different ways, for example by intravenous, intraperitoneal,subcutaneous, intracranial, intrathecal, intraarterial (e.g., viacarotid), and intramuscular administration.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, and alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, or fixed oils. Intravenousvehicles include liquid and nutrient supplements, electrolytesupplements and the like. Preservatives and other additives may also bepresent, for example, such as antimicrobial agents, antioxidants,chelating agents and inert gases.

The medical staff will determine the dosage regimen based on variousclinical factors. As is well known in the medical field, the dosage ofany patient depends on a variety of factors, including the patient'ssize, body surface area, age, the specific compound to be administered,sex, frequency and route of administration, overall health and otherdrugs administered simultaneously. The dosage range of thepharmaceutical composition comprising plasminogen of the presentinvention may be, for example, such as about 0.0001 to 2000 mg/kg, orabout 0.001 to 500 mg/kg (such as 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg,0.75 mg/kg, 10 mg/kg and 50 mg/kg) of the subject's body weight daily.For example, the dosage may be 1 mg/kg body weight or 50 mg/kg bodyweight, or in the range of 1 mg/kg-50 mg/kg, or at least 1 mg/kg.Dosages above or below this exemplary range are also contemplated,especially considering the above factors. The intermediate dosages inthe above range are also included in the scope of the present invention.A subject may be administered with such dosages daily, every other day,weekly or based on any other schedule determined by empirical analysis.An exemplary dosage schedule includes 1-10 mg/kg for consecutive days.During administration of the drug of the present invention, thetherapeutic effect and safety are required to be assessed real-timely.

Articles of Manufacture or Kits

One embodiment of the present invention relates to an article ofmanufacture or a kit comprising plasminogen of the present invention orplasmin useful in the treatment of angiocardiopathy and its relatedconditions caused by diabetes mellitus. The article preferably includesa container, label or package insert. Suitable containers includebottles, vials, syringes and the like. The container can be made ofvarious materials, such as glass or plastic. The container contains acomposition that is effective to treat the disease or condition of thepresent invention and has a sterile access (for example, the containermay be an intravenous solution bag or vial containing a plug that can bepierced by a hypodermic injection needle). At least one active agent inthe composition is plasminogen/plasmin. The label on or attached to thecontainer indicates that the composition is used to treat theangiocardiopathy and its related conditions caused by diabetes mellitusaccording to the present invention. The article may further comprise asecond container containing a pharmaceutically acceptable buffer, suchas phosphate buffered saline, Ringer's solution and glucose solution. Itmay further comprise other substances required from a commercial anduser perspective, including other buffers, diluents, filters, needlesand syringes. In addition, the article comprises a package insert withinstructions for use, including, for example, instructions to direct auser of the composition to administer to a patient the plasminogencomposition and other drugs for treating an accompanying disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows detection results of serum high-density lipoproteincholesterol after administration of plasminogen to 3% cholesterolhyperlipemia model mice for 10 days and 20 days. The results showed thatthe concentration of HDL-C in serum of mice in the group administeredwith plasminogen was remarkably higher than that in the control groupadministered with vehicle PBS, and the high-density lipoproteinconcentrations of the two groups were statistically different afteradministration for 10 or 20 days (** indicates P<0.01). It indicatesthat plasminogen can effectively elevate the content of high-densitylipoprotein cholesterol in serum of hyperlipemia model mice, and improvethe dyslipidemia in hyperlipemia model mice.

FIG. 2 shows detection results of serum total cholesterol afteradministration of plasminogen to 3% cholesterol hyperlipemia model micefor 20 days. The results showed that the concentration of totalcholesterol in mice in the group administered with plasminogen wasremarkably lower than that in the control group administered withvehicle PBS, and the statistical difference was significant (* indicatesP<0.05). It indicates that plasminogen can lower the content of serumtotal cholesterol in hyperlipemia model mice, and has an effect oflowering blood lipid.

FIG. 3 shows detection results of serum low-density lipoproteincholesterol after administration of plasminogen to 3% cholesterolhyperlipemia model mice for 20 days. The results showed that theconcentration of LDL-C in mice in the group administered withplasminogen was remarkably lower than that in the control groupadministered with vehicle PBS, and the statistical difference wassignificant (* indicates P<0.05). It indicates that plasminogen canlower the content of low-density lipoprotein cholesterol in serum ofhyperlipemia model mice, and has an effect of improving hyperlipemia.

FIG. 4 shows observed results of oil red 0 staining of liver afteradministration of plasminogen to 16-week hyperlipemia model mice for 30days. A represents the control group administered with vehicle PBS, Brepresents the group administered with plasminogen, and C represents thequantitative analysis results. The results showed that the fatdeposition in liver of mice in the group administered with plasminogenwas remarkably lower than that in the control group administered withvehicle PBS, and the quantitative analysis showed significantstatistical difference (* indicates P<0.05). It indicates thatplasminogen can ameliorate fat deposition in liver of hyperlipemia modelmice.

FIG. 5 shows observed results of oil red 0 staining of aortic sinusafter administration of plasminogen to 16-week hyperlipemia model micefor 30 days. A and C represent the control group administered withvehicle PBS, B and D represent the group administered with plasminogen,and E represents the quantitative analysis results. The results showedthat the fat deposition in aortic sinus of mice in the groupadministered with plasminogen was remarkably lower than that in thecontrol group administered with vehicle PBS, and the statisticaldifference was significant (* indicates P<0.05). It indicates thatplasminogen can ameliorate fat deposition in aortic sinus ofhyperlipemia model mice.

FIG. 6 shows a representative image of HE staining of aortic sinus afteradministration of plasminogen to 16-week hyperlipemia model mice for 30days. A and C refer to the control group administered with vehicle PBS,and B and D refer to the group administered with plasminogen. Theresults showed that the foam cell deposition (indicated by arrow) andthe plaque deposition on the aortic wall in the control groupadministered with vehicle PBS were severe; while in the groupadministered with plasminogen, only a mild foam cell deposition wasobserved on the aortic wall, no obvious atherosclerotic plaquedeposition was observed under the intima, and the aortic injury in thegroup administered with plasminogen was relatively minor. It indicatesthat plasminogen can ameliorate the injury caused by lipid deposition onthe inner wall of aortic sinus of hyperlipemia model mice.

FIG. 7 shows an image of immunohistochemical staining of cardiac fibrinafter administration of plasminogen to 16-week hyperlipemia model micefor 30 days. A represents the control group administered with vehiclePBS, B represents the group administered with plasminogen, and Crepresents the quantitative analysis results. The results showed thatthe positive expression of cardiac fibrin in mice in the groupadministered with plasminogen was remarkably lower than that in thecontrol group administered with vehicle PBS, and the statisticaldifference was significant (* indicates P<0.05). It indicates thatplasminogen can reduce the cardiac injury caused by hyperlipemia.

FIG. 8 shows a representative image of IgM immunostaining of heart afteradministration of plasminogen to 16-week hyperlipemia model mice for 30days. A represents the control group administered with vehicle PBS, andB represents the group administered with plasminogen. The results showedthat the positive expression of IgM in the heart of mice in the groupadministered with plasminogen was remarkably less than that in thecontrol group administered with vehicle PBS, indicating that plasminogencan alleviate the cardiac injury caused by hyperlipemia.

FIG. 9 shows a representative image of Sirius red staining of heartafter administration of plasminogen to 16-week hyperlipemia model micefor 30 days. A represents the control group administered with vehiclePBS, and B represents the group administered with plasminogen. Theresults showed that the collagen deposition in the group administeredwith plasminogen was remarkably less than that in the control groupadministered with vehicle PBS, indicating that plasminogen can alleviatethe cardiac fibrosis in hyperlipemia model mice.

FIG. 10 shows detection results of serum troponin after administrationof plasminogen to 16-week hyperlipemia model mice for 30 days. Theresults showed that the concentration of cardiac troponin in serum inthe control group administered with vehicle PBS was remarkably higherthan that in the group administered with plasminogen, and thestatistical difference was significant (* indicates P<0.05). Itindicates that plasminogen can significantly repair the damage tohyperlipidemic heart.

FIG. 11 shows detection results of serum atherosclerosis index afteradministration of plasminogen to 3% cholesterol hyperlipemia model micefor 20 days. The results showed that the atherosclerosis index of micein the group administered with plasminogen was remarkably lower thanthat in the control group administered with vehicle PBS, and thestatistical difference was extremely significant (** indicates P<0.01).It indicates that plasminogen can effectively lower the risk ofatherosclerosis in hyperlipemia model mice.

FIG. 12 shows results of serum cardiac risk index after administrationof plasminogen to 3% cholesterol hyperlipemia model mice for 20 days.The results showed that CRI in the group administered with plasminogenwas remarkably lower than that in the control group administered withvehicle PBS, and the statistical difference was extremely significant(** indicates P<0.01). It indicates that plasminogen can effectivelylower the risk of heart disease in hyperlipemia model mice.

FIG. 13 shows an image of oil red 0 staining of liver afteradministration of plasminogen to 24- to 25-week diabetic mice for 35days. The results showed that the lipid deposition area in liver of micein the group administered with plasminogen was significantly less thanthat in the control group administered with vehicle PBS, and thestatistical difference was significant (* indicates P<0.05). Itindicates that plasminogen can reduce fat deposition in liver ofdiabetic mice.

FIG. 14 shows an image of HE staining of aorta after administration ofplasminogen to 24- to 25-week-old diabetic mice for 31 days. A and Crefer to the control group administered with vehicle PBS, and B and Drefer to the group administered with plasminogen. The results showedthat in the control group administered with vehicle PBS, there was afoam cell deposition (indicated by arrow) on the vascular wall, themiddle elastic membrane was arranged in disorder, and the vascular wallwas thickened and accidented; while in the group administered withplasminogen, the middle elastic membrane had a regular structure in awave shape, and the thickness of vascular wall was uniform. It indicatesthat the injection of plasminogen has a certain repair effect on aorticinjury caused by diabetes mellitus.

FIG. 15 shows a representative image of oil red 0 staining of ventricleafter administration of plasminogen to 26-week-old diabetic mice for 35days. A represents the control group administered with vehicle PBS, andB represents the group administered with plasminogen. The results showedthat the lipid deposition in ventricle (indicated by arrow) of mice inthe group administered with plasminogen was remarkably less than that inthe control group administered with vehicle PBS. It indicates thatplasminogen can reduce lipid deposition in ventricle of diabetic mice,and promote the repair of ventricular injury.

FIG. 16 shows detection results of the content of high-densitylipoprotein cholesterol in serum after administration of plasminogen to26-week-old diabetic mice for 35 days. The results showed that after 35days of continuous injection of human plasminogen into diabetic mice,the content of HDL-C in serum of mice in the group administered withplasminogen was higher than that in the control group administered withvehicle PBS, and the statistical difference was significant (* indicatesP<0.05). It indicates that the injection of plasminogen can promote theincrease in the content of serum high-density lipoprotein cholesterol,and improve the dyslipidemia in diabetic mice.

FIG. 17 shows detection results of the content of low-densitylipoprotein cholesterol (LDL-C) in serum after administration ofplasminogen to 24- to 25-week-old diabetic mice for 31 days. The resultsshowed that after continuous injection of human plasminogen intodiabetic model mice for 31 days, the content of LDL-C in serum of micein the group administered with plasminogen was lower than that in thecontrol group administered with vehicle PBS, and the statisticaldifference was close to significant (P=0.1). It indicates thatplasminogen can lower the content of low-density lipoprotein cholesterolin serum of diabetic mice.

FIG. 18 shows detection results of serum total cholesterol afteradministration of plasminogen to ApoE atherosclerosis model mice for 30days. The results showed that the concentration of total cholesterol inmice in the group administered with plasminogen was remarkably lowerthan that in the control group administered with vehicle PBS, and thestatistical difference was significant (* indicates P<0.05). Itindicates that plasminogen can lower the content of total cholesterol inserum of ApoE atherosclerosis model mice, and improve the dyslipidemiain atherosclerosis model mice.

FIG. 19 shows detection results of serum triglyceride afteradministration of plasminogen to ApoE atherosclerosis model mice for 30days. The results showed that the concentration of triglyceride in micein the group administered with plasminogen was remarkably lower thanthat in the control group administered with vehicle PBS, and thestatistical difference was significant (* indicates P<0.05). Itindicates that plasminogen can lower the content of triglyceride inserum of ApoE atherosclerosis model mice, and improve the dyslipidemiain atherosclerosis model mice.

FIG. 20 shows detection results of serum low-density lipoproteincholesterol after administration of plasminogen to ApoE atherosclerosismodel mice for 30 days. The results showed that the concentration ofLDL-C in mice in the group administered with plasminogen was remarkablylower than that in the control group administered with vehicle PBS, andthe statistical difference was significant (* indicates P<0.05). Itindicates that plasminogen can lower the content of low-densitylipoprotein cholesterol in serum of ApoE atherosclerosis model mice, andimprove the dyslipidemia in atherosclerosis model mice.

FIG. 21 shows a representative image of oil red O staining of liverafter administration of plasminogen to ApoE atherosclerosis model micefor 30 days. A represents the control group administered with vehiclePBS, B represents the group administered with plasminogen, and Crepresents the quantitative analysis results. The results showed thatthe fat deposition in liver of mice in the group administered withplasminogen was remarkably lower than that in the control groupadministered with vehicle PBS, and the quantitative analysis showedsignificant statistical difference (* indicates P<0.05). It indicatesthat plasminogen can reduce fat deposition in liver of atherosclerosismodel mice.

FIG. 22 shows a representative image of oil red O staining of aorticsinus after administration of plasminogen to ApoE atherosclerosis modelmice for 30 days. A represents the control group administered withvehicle PBS, and B represents the group administered with plasminogen.The results showed that the fat deposition in aortic sinus of mice inthe group administered with plasminogen was remarkably lower than thatin the control group administered with vehicle PBS. It indicates thatplasminogen can ameliorate fat deposition in aortic sinus ofatherosclerosis model mice.

FIG. 23 shows a representative image of Sirius red staining of aorticsinus after administration of plasminogen to 16-week-old hyperlipemiamodel mice for 30 days. A and C refer to the control group administeredwith vehicle PBS, and B and D refer to the group administered withplasminogen. The results showed that the area of collagen deposition(indicated by arrow) on the inner walls of blood vessels of aortic sinusin the group administered with plasminogen was remarkably less than thatin the control group administered with vehicle PBS, indicating thatplasminogen can alleviate the level of aortic sinus fibrosis inhyperlipemia model mice.

FIG. 24 shows statistical results of cardiac coefficient afteradministration of plasminogen to ApoE atherosclerosis model mice for 30days. The results showed that the cardiac organ coefficient of mice inthe group administered with plasminogen was remarkably lower than thatin the control group administered with vehicle PBS. It indicates thatplasminogen can ameliorate the compensatory cardiac hypertrophy causedby cardiac injury in ApoE atherosclerosis model mice.

FIG. 25 shows observed results of Sirius red staining of kidney afteradministration of plasminogen to 3% cholesterol hyperlipemia model micefor 30 days. A represents the blank control group, B represents thecontrol group administered with vehicle PBS, C represents the groupadministered with plasminogen, and D represents the quantitativeanalysis results. The results showed that the collagen deposition inkidney (indicated by arrow) in the group administered with plasminogenwas remarkably less than that in the control group administered withvehicle PBS, and the statistical difference was significant; and in thegroup administered with plasminogen, fibrosis was substantially restoredto a normal level. It indicates that plasminogen can effectively reducerenal fibrosis in 3% cholesterol hyperlipemia model mice.

FIG. 26 shows observed results of oil red 0 of kidney afteradministration of plasminogen to 3% cholesterol hyperlipemia model micefor 30 days. A represents the blank control group, B represents thecontrol group administered with vehicle PBS, C represents the groupadministered with plasminogen, and D represents the quantitativeanalysis results. The results showed that the fat deposition in kidney(indicated by arrow) of mice in the group administered with plasminogenwas remarkably less than that in the control group administered withvehicle PBS, and the quantitative analysis showed significantstatistical difference; in addition, the lipid deposition level in thegroup administered with plasminogen was similar to that in mice in theblank control group. It indicates that plasminogen can reduce the fatdeposition in kidney of hyperlipemia model mice, and thus reduce renalinjury caused by fat deposition.

EXAMPLES Example 1 Plasminogen Increases the Concentration of SerumHigh-Density Lipoprotein Cholesterol in 3% Cholesterol HyperlipemiaModel Mice

Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fatdiet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia^([30,31]).This model was designated as the 3% cholesterol hyperlipemia model. Themodel mice continued to be fed with a 3% cholesterol high-fat diet. 50μL of blood was taken from each mouse three days before administration,and the total cholesterol was detected. The mice were randomly dividedinto two groups based on the total cholesterol concentration and thebody weight, 8 mice in each group. The first day of administration wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day viathe tail vein, and an equal volume of PBS was administered to mice inthe control group administered with vehicle PBS via the tail vein, bothlasting for 20 days. On Day 10 and Day 20, the mice fasted for 16 hours,and on Day 11 and Day 21, 50 μL of blood was collected from orbitalvenous plexus, and centrifuged to obtain a supernatant, which was usedin detecting the serum high-density lipoprotein cholesterol (HDL-C). Thecontent of high-density lipoprotein cholesterol herein was detected bythe method as described in a detection kit (Nanjing JianchengBioengineering Institute, Cat# A112-1).

High-density lipoprotein is an anti-atherosclerosisplasma lipoprotein, aprotective factor of coronary heart disease, commonly known as “vascularscavenger”.

The detection results showed that the concentration of HDL-C in serum ofmice in the group administered with plasminogen was remarkably higherthan that in the control group administered with vehicle PBS, and theHDL-C concentrations of the two groups were statistically differentafter administration for 10 or 20 days (FIG. 1). It indicates thatplasminogen can elevate the content of high-density lipoproteincholesterol in serum of hyperlipemia model mice, and improve thedyslipidemia in mice with hyperlipemia.

Example 2 Plasminogen Lowers the Serum Total Cholesterol Level in 3%Cholesterol Hyperlipemia Model Mice

Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fatdiet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia^([30,31]).This model was designated as the 3% cholesterol hyperlipemia model. Themodel mice continued to be fed with a 3% cholesterol high-fat diet. 50μL of blood was taken from each mouse three days before administration,and the total cholesterol was detected. The mice were randomly dividedinto two groups based on the total cholesterol concentration and thebody weight, 8 mice in each group. The first day of administration wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day viathe tail vein, and an equal volume of PBS was administered to mice inthe control group administered with vehicle PBS via the tail vein, bothlasting for 20 days. On Day 20, the mice fasted for 16 hours, and on Day21, 50 μL of blood was collected from orbital venous plexus, andcentrifuged to obtain a supernatant. The total cholesterol was detectedusing a total cholesterol detection kit (Nanjing JianchengBioengineering Institute, Cat# A111-1).

The detection results showed that the concentration of total cholesterolin mice in the group administered with plasminogen was remarkably lowerthan that in the control group administered with vehicle PBS, and thestatistical difference was significant (FIG. 2). It indicates thatplasminogen can lower the content of serum total cholesterol inhyperlipemia model mice.

Example 3 Plasminogen Lowers the Serum Low-Density LipoproteinCholesterol Level in 3% Cholesterol Hyperlipemia Model Mice

Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fatdiet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia^([30,31]).This model was designated as the 3% cholesterol hyperlipemia model. Themodel mice continued to be fed with a 3% cholesterol high-fat diet. 50μL of blood was taken from each mouse three days before administration,and the total cholesterol was detected. The mice were randomly dividedinto two groups based on the total cholesterol concentration and thebody weight, 8 mice in each group. The first day of administration wasrecorded as Day 1. Mice in the group administered with plasminogen wereinjected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day viathe tail vein, and an equal volume of PBS was administered to mice inthe control group administered with vehicle PBS via the tail vein, bothlasting for 20 days. On Day 20, the mice fasted for 16 hours, and on Day21, 50 μL of blood was collected from orbital venous plexus, andcentrifuged to obtain a supernatant. The low-density lipoproteincholesterol (LDL-C) was detected using a low-density lipoproteincholesterol detection kit (Nanjing Jiancheng Bioengineering Institute,Cat# A113-1).

Low-density lipoprotein is a lipoprotein particle that carriescholesterol into peripheral tissue cells and can be oxidized intooxidized low-density lipoprotein. When low-density lipoprotein,particularly oxidized low-density lipoprotein (OX-LDL) is in excess, thecholesterol it carries accumulates on the arterial wall, causingarteriosclerosis. Therefore, low-density lipoprotein cholesterol iscalled “bad cholesterol”.

The results showed that the concentration of LDL-C in mice in the groupadministered with plasminogen was remarkably lower than that in thecontrol group administered with vehicle PBS, and the statisticaldifference was significant (FIG. 3). It indicates that plasminogen canreduce the content of low-density lipoprotein cholesterol in serum ofhyperlipemia model mice, and improve the dyslipidemia in mice withhyperlipemia.

Example 4 Plasminogen Reduces the Fat Deposition in Liver of 16-WeekHyperlipemia Model Mice

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([30,31]). This model was designated as the16-week hyperlipemia model. The model mice continued to be fed with ahigh-cholesterol diet. 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol (T-CHO) contentwas detected. The mice were randomly divided into two groups based onthe T-CHO content, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The mice were administered for 30 daysand sacrificed on Day 31. The livers were fixed in 4% paraformaldehydefor 24 to 48 hours, then sedimented in 15% and 30% sucrose at 4° C.overnight, respectively, and embedded in OCT. The frozen sections were 8μm thick, stained with oil red 0 for 15 min, differentiated with 75%ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s,and sealing with glycerine and gelatin. The sections were observed underan optical microscope at 200×.

Oil red O staining can show lipid deposition and reflect the extent oflipid deposition^([32]). The results showed that the fat deposition inliver of mice in the group administered with plasminogen (FIG. 4B) wasremarkably lower than that in the control group administered withvehicle PBS (FIG. 4A), and the quantitative analysis showed significantstatistical difference (FIG. 4C). It indicates that plasminogen canreduce fat deposition in liver of hyperlipemia model mice.

Example 5 Plasminogen Reduces Lipid Deposition in Aortic Sinus of16-Week Hyperlipemia Model Mice

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([30,31]). This model was designated as the16-week hyperlipemia model. The model mice continued to be fed with ahigh-cholesterol diet. 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol (T-CHO) contentwas detected. The mice were randomly divided into two groups based onthe T-CHO content, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The mice were administered for 30 daysand sacrificed on Day 31. The heart tissues were fixed in 4%paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30%sucrose at 4° C. overnight, respectively, and embedded in OCT. Thefrozen sections of aortic sinus were 8 μm thick, stained with oil red 0for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclearstaining with hematoxylin for 30 s, and sealing with glycerine andgelatin. The sections were observed under an optical microscope at 40×(FIGS. 5A and 5B) and 200× (FIGS. 5C and 5D).

The results showed that the fat deposition in aortic sinus of mice inthe group administered with plasminogen (FIGS. 5B and 5D) was remarkablylower than that in the control group administered with vehicle PBS(FIGS. 5A and 5C), and the statistical difference was significant (FIG.5E). It indicates that plasminogen can reduce lipid deposition in aorticsinus of hyperlipemia model mice.

Example 6 Plasminogen Improves Aortic Sinus Injury in 16-WeekHyperlipemia Model Mice

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([30,31]). This model was designated as the16-week hyperlipemia model. The model mice continued to be fed with ahigh-cholesterol diet. 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol (T-CHO) contentwas detected. The mice were randomly divided into two groups based onthe T-CHO content, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The mice were administered for 30 daysand sacrificed on Day 31. The heart tissues were fixed in 4%paraformaldehyde for 24 to 48 hours. The fixed tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The fixed tissue samples wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The aortic sinus tissue sections were 3 μmthick. The sections were dewaxed and rehydrated, stained withhematoxylin and eosin (HE staining), differentiated with 1% hydrochloricacid in alcohol, and returned to blue with ammonia water. The sectionswere sealed after dehydration with alcohol gradient, and observed underan optical microscope at 40× (FIGS. 6A and B) and 400× (FIGS. 6C and D).

The results showed that the foam cell deposition (indicated by arrow)and the plaque deposition on the inner wall of aortic sinus in thecontrol group administered with vehicle PBS (FIGS. 6A and C) weresevere; while in the group administered with plasminogen (FIGS. 6B andD), only a mild foam cell deposition was observed on the inner wall ofaortic sinus, no obvious atherosclerotic plaque deposition was observedunder the intima, and the injury to the inner wall of aortic sinus inthe group administered with plasminogen was relatively minor. Itindicates that plasminogen can ameliorate the damage to the inner wallof arterial sinus of hyperlipemia model mice.

Example 7 Plasminogen Reduces Expression of Cardiac Fibrin in 16-WeekHyperlipemia Model Mice

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([30,31]). This model was designated as the16-week hyperlipemia model. The model mice continued to be fed with ahigh-cholesterol diet. 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol (T-CHO) contentwas detected. The mice were randomly divided into two groups based onthe T-CHO content, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The mice were administered for 30 daysand sacrificed on Day 31. The heart tissues were fixed in 4%paraformaldehyde for 24 to 48 hours. The fixed tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The sections were incubated with 3% hydrogen peroxide for 15 minutes andwashed with water twice for 5 minutes each time. The sections wereblocked with 5% normal goat serum liquid (Vector laboratories, Inc.,USA) for 30 minutes, and after the time was up, the goat serum liquidwas discarded, and the tissues were circled with a PAP pen. The sectionswere incubated with 3% hydrogen peroxide for 15 minutes and washed withwater twice for 5 minutes each time. The sections were incubated withrabbit anti-mouse fibrin antibody (Abcam) overnight at 4° C. and washedwith 0.01M PBS twice for 5 minutes each time. The sections wereincubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody(Abcam), for 1 hour at room temperature and washed with PBS twice for 5minutes each time. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washed with water three times, thesections were counterstained with hematoxylin for 30 seconds and flushedwith running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylenehe, and sealing with a neutralgum, the sections were observed under an optical microscope at 200×.

Fibrinogen is the precursor of fibrin, and in the presence of tissueinjury, as a stress response to the body's injury, fibrinogen ishydrolyzed into fibrin and deposited at the injury site^([33,34]).Therefore, the local fibrin level at the injury site can be used as asign of the degree of injury.

The immunohistochemical staining results showed that the positiveexpression of cardiac fibrin in mice in the group administered withplasminogen (FIG. 7B) was remarkably less than that in the control groupadministered with vehicle PBS (FIG. 7A), and the statistical differencewas significant (FIG. 7C), indicating that plasminogen can reduce amyocardial injury caused by hyperlipemia.

Example 8 Plasminogen Protects 16-Week Hyperlipemia Model Mice fromMyocardial Injury Effectively

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([30,31]). This model was designated as the16-week hyperlipemia model. The model mice continued to be fed with ahigh-cholesterol diet. 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol (T-CHO) contentwas detected. The mice were randomly divided into two groups based onthe T-CHO content, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The mice were administered for 30 daysand sacrificed on Day 31. The heart tissues were fixed in 4%paraformaldehyde for 24 to 48 hours. The fixed tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The sections were incubated with 3% hydrogen peroxide for 15 minutes andwashed with water twice for 5 minutes each time. The sections wereblocked with 5% normal goat serum liquid (Vector laboratories, Inc.,USA) for 30 minutes, and after the time was up, the goat serum liquidwas discarded, and the tissues were circled with a PAP pen. The sectionswere incubated with 3% hydrogen peroxide for 15 minutes and washed withwater twice for 5 minutes each time. The sections were incubated withgoat anti-mouse IgM (HRP) antibody (Abcam) for 1 hour at roomtemperature and washed with PBS twice for 5 minutes each time. Thesections were developed with a DAB kit (Vector laboratories, Inc., USA).After washed with water three times, the sections were subjected tonuclear staining with hematoxylin for 30 seconds and flushing withrunning water for 5 minutes. After dehydration with alcohol gradient,permeabilization with xylenehe, and sealing with a neutral gum, thesections were observed under an optical microscope at 200×.

IgM antibodies play an important role during the clearance of apoptoticand necrotic cells, and the local level of IgM antibodies in damagedtissues and organs is positively correlated with the degree ofinjury¹³⁵′³⁶¹. Therefore, detection of local level of IgM antibodies intissues and organs can reflect the extent of injury of the tissues andorgans.

The immunostaining results showed that the positive expression of IgM inthe heart of mice in the group administered with plasminogen (FIG. 8B)was remarkably less than that in the control group administered withvehicle PBS (FIG. 8A), indicating that plasminogen can reduce thecardiac injury in hyperlipemia model animals.

Example 9 Plasminogen Alleviates Cardiac Fibrosis in 16-WeekHyperlipemia Model Mice

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([30,31]). This model was designated as the16-week hyperlipemia model. The model mice continued to be fed with ahigh-cholesterol diet. 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol (T-CHO) contentwas detected. The mice were randomly divided into two groups based onthe T-CHO content, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The mice were administered for 30 daysand sacrificed on Day 31. The heart tissues were fixed in 4%paraformaldehyde for 24 to 48 hours. The fixed tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The tissue sections was 3 μm thick. Thesections were dewaxed and rehydrated and washed with water once. Afterstained with 0.1% Sirius red in saturated picric acid for 30 min, thesections were flushed with running water for 2 min. After stained withhematoxylin for 1 min, the sections were flushed with running water,differentiated with 1% hydrochloric acid in alcohol, returned to bluewith ammonia water, flushed with running water, dried and sealed with aneutral gum. The sections were observed under an optical microscope at200×.

Sirius red staining allows for long-lasting staining of collagen. As aspecial staining method for pathological sections, Sirius red stainingcan show the collagen tissue specifically.

The staining results showed that the deposition of collagen in the groupadministered with plasminogen (FIG. 9B) was remarkably less than that inthe control group administered with vehicle PBS (FIG. 9A), indicatingthat plasminogen can reduce the deposition of collagen in the hearttissues of hyperlipemia model mice and alleviate myocardial fibrosis.

Example 10 Plasminogen Repairs Myocardial Injury in 16-Week HyperlipemiaModel Mice

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([30,31]). This model was designated as the16-week hyperlipemia model. The model mice continued to be fed with ahigh-cholesterol diet. 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol (T-CHO) contentwas detected. The mice were randomly divided into two groups based onthe T-CHO content, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The administration lasted for 30 days.After administration on Day 30, the mice began to fast for 16 hours, andon Day 31, the blood was collected from removed eyeballs, andcentrifuged to obtain a supernatant, which was detected for theconcentration of troponin in serum using cardiac troponin (Cardiactroponin I, CTNI) detection kit (Nanjing Jiancheng).

Cardiac troponin I is an important marker of myocardial injury, and itsserum concentration can reflect the extent of myocardial injury^([37]).

The detection results showed that the concentration of cardiac troponinin serum in the control group administered with vehicle PBS wasremarkably higher than that in the group administered with plasminogen,and the statistical difference was significant (FIG. 10). It indicatesthat plasminogen can significantly ameliorate the cardiac injury inhyperlipemia model mice.

Example 11 Plasminogen Lowers Risk of Atherosclerosis Formation in 3%Cholesterol Hyperlipemia Model Mice

Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fatdiet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia^([30,31]).This model was designated as the 3% cholesterol hyperlipemia model. Themodel mice continued to be fed with a 3% cholesterol high-fat diet. 50μL of blood was taken from each mouse three days before administration,and the total cholesterol (T-CHO) was detected. The mice were randomlydivided into two groups based on the total cholesterol concentration andthe body weight, 8 mice in each group. The first day of administrationwas recorded as Day 1. Mice in the group administered with plasminogenwere injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/dayvia the tail vein, and an equal volume of PBS was administered to micein the control group administered with vehicle PBS via the tail vein.After administration on Day 20, the mice began to fast for 16 hours, andon Day 21, 50 μL of blood was collected from orbital venous plexus, andcentrifuged to obtain a supernatant. The total cholesterol content wasdetected by using a total cholesterol detection kit (Nanjing JianchengBioengineering Institute, Cat# A111-1); and the high-density lipoproteincholesterol (HDL-C) content was detected using a high-densitylipoprotein cholesterol detection kit (Nanjing Jiancheng BioengineeringInstitute, Cat# A112-1).

Atherosclerosis index is a comprehensive index to predictatherosclerosis clinically. It is considered to be of greater clinicalimportance as an estimate of the risk of coronary heart disease thantotal cholesterol, triglyceride, high-density lipoprotein, andlow-density lipoprotein alone^([38]). Atherosclerosisindex=(T-CHO-HDL-C)/HDL-C.

The calculation results showed that the atherosclerosis index of mice inthe group administered with plasminogen was remarkably lower than thatin the control group administered with vehicle PBS, and the statisticaldifference was significant (FIG. 11). It indicates that plasminogen canlower the risk of atherosclerosis in hyperlipemia model mice.

Example 12 Plasminogen Lowers Risk of Onset of Heart Disease in 3%Cholesterol Hyperlipemia Model Mice

Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fatdiet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia^([30,31]).This model was designated as the 3% cholesterol hyperlipemia model. Themodel mice continued to be fed with a 3% cholesterol high-fat diet. 50μL of blood was taken from each mouse three days before administration,and the total cholesterol (T-CHO) was detected. The mice were randomlydivided into two groups based on the total cholesterol concentration, 8mice in each group. The first day of administration was recorded asDay 1. Mice in the group administered with plasminogen were injectedwith human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tailvein, and an equal volume of PBS was administered to mice in the controlgroup administered with vehicle PBS via the tail vein. Afteradministration on Day 20, the mice began to fast for 16 hours, and onDay 21, 50 μL of blood was collected from orbital venous plexus, andcentrifuged to obtain a supernatant. The total cholesterol content wasdetected by using a total cholesterol detection kit (Nanjing JianchengBioengineering Institute, Cat# A111-1); and the high-density lipoproteincholesterol (HDL-C) content was detected using a high-densitylipoprotein cholesterol detection kit (Nanjing Jiancheng BioengineeringInstitute, Cat# A112-1). Cardiac risk index=T-CHO/HDL-C.

Cardiac risk index (CRI) is used to assess the risk of heart diseaseinduced by dyslipidemia^([38]).

The results showed that CRI in the group administered with plasminogenwas remarkably lower than that in the control group administered withvehicle PBS, and the statistical difference was extremely significant(FIG. 12). It indicates that plasminogen can effectively lower the riskof heart disease in hyperlipemia model mice.

Example 13 Plasminogen Ameliorates Lipid Deposition in Liver of DiabeticMice

Ten 24- to 25-week-old male db/db mice were randomly divided into twogroups, five in the control group administered with vehicle PBS and fivein the group administered with plasminogen, respectively. The mice wereweighed and grouped on the day when the experiment began, i.e. day 0.Plasminogen or PBS was administered from day 1. Mice in the groupadministered with plasminogen were injected with plasminogen at a doseof 2 mg/0.2 mL/mouse/day via the tail vein, and an equal volume of PBSwas administered to mice in the control group administered with vehiclePBS via the tail vein, both lasting for 35 consecutive days. The micewere sacrificed on Day 36. The liver tissues were fixed in 4%paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30%sucrose at 4° C. overnight, respectively, and embedded in OCT. Thefrozen sections were 8 μm thick, stained with oil red 0 for 15 min,differentiated with 75% ethanol for 5 s followed by nuclear stainingwith hematoxylin for 30 s, and sealing with glycerine and gelatin. Thesections were observed under an optical microscope at 200×.

The staining results showed that the lipid deposition area in liver ofmice in the group administered with plasminogen (FIG. 13B) wassignificantly lower than that in the control group administered withvehicle PBS (FIG. 13A), and the statistical difference was significant(P=0.02) (FIG. 13C). It indicates that plasminogen can reduce fatdeposition in liver of diabetic mice.

Example 14 Plasminogen Alleviates Injury of Aortic Wall in Diabetic Mice

Ten 24- to 25-week-old male db/db mice were randomly divided into twogroups, five in the control group administered with vehicle PBS and fivein the group administered with plasminogen, respectively. The mice wereweighed and grouped on the day when the experiment began, i.e. Day 0.PBS or plasminogen was administered from day 1 for 31 consecutive days.Mice in the group administered with plasminogen were injected withplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein. Mice were sacrificed onDay 32, and the aortas were fixed in 10% neutral formalin fixative for24 hours. The fixed aortas were paraffin-embedded after dehydration withalcohol gradient and permeabilization with xylene. The tissue sectionswere 5 μm thick. The sections were dewaxed and rehydrated, stained withhematoxylin and eosin (HE staining), differentiated with 1% hydrochloricacid in alcohol, and returned to blue with ammonia water. The sectionswere sealed after dehydration with alcohol gradient, and observed underan optical microscope at 400× (FIGS. 14A and B) and at 1000× (FIGS. 14Cand D) oil immersion lens.

Diabetes mellitus with hyperlipemia is a common complication of diabetesmellitus and an important risk factor for diabeticmacroangiopathyl^([39]).

The staining results showed that in the control group administered withvehicle PBS (FIGS. 14A and C), there was a foam cell deposition(indicated by arrow) on the vascular wall, the middle elastic membranewas arranged in disorder, and the vascular wall was thickened andaccidented; while in the group administered with plasminogen (FIGS. 14Band D), the middle elastic membrane has a regular structure in a waveshape, and the thickness of vascular wall was uniform. It indicates thatthe injection of plasminogen can reduce lipid deposition on the aorticwall of diabetic mice, and has a certain protective effect on the injurycaused by lipid deposition on the arterial wall.

Example 15 Plasminogen Lowers Lipid Deposition in Ventricle of DiabeticMice

Nine 26-week-old male db/db mice were randomly divided into groups, 4mice in the group administered with plasminogen, and 5 mice in thecontrol group administered with vehicle PBS. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 2 mg/0.2 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein, both lasting for 35 days. The mice weresacrificed on Day 36. The hearts were fixed in 4% paraformaldehyde for24 to 48 hours, then sedimented in 15% and 30% sucrose at 4° C.overnight, respectively, and embedded in OCT. The frozen sections were 8μm thick, stained with oil red 0 for 15 min, differentiated with 75%ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s,and sealing with glycerine and gelatin. The sections were observed underan optical microscope at 400×.

The results showed that the lipid deposition in ventricle (indicated byarrow) of mice in the group administered with plasminogen (FIG. 15B) wasremarkably less than that in the control group administered with vehiclePBS (FIG. 15A). It indicates that plasminogen can reduce fat depositionin ventricle of diabetic mice, and promote the repair of ventricularinjury.

Example 16 Plasminogen Increases the High-Density LipoproteinCholesterol Level in Serum of Diabetic Mice

Twenty 26-week-old male db/db mice were randomly divided into groups, 11mice in the group administered with plasminogen, and 9 mice in thecontrol group administered with vehicle PBS. The mice were weighed andgrouped on the day when the experiment began, i.e. Day 0. Plasminogen orPBS was administered from day 1 for 35 consecutive days. Mice in thegroup administered with plasminogen were injected with human plasminogenat a dose of 2 mg/0.2 mL/mouse/day via the tail vein, and an equalvolume of PBS was administered to mice in the control group via the tailvein. On Day 36, the whole blood was collected from removed eyeballs inmice, and centrifuged at 3500 r/min at 4° C. for 10 min to obtain asupernatant, which was detected for the concentration of high-densitylipoprotein cholesterol (HDL-C) in serum using a high-densitylipoprotein detection kit (Nanjing Jiancheng Bioengineering Institute,Cat# A112-1).

The detection results showed that the content of HDL-C in serum of micein the group administered with plasminogen was higher than that in thecontrol group administered with vehicle PBS, and the statisticaldifference was significant (FIG. 16). It indicates that the injection ofplasminogen can promote the increase in the content of serumhigh-density lipoprotein cholesterol, and improve the dyslipidemia ofdiabetes mellitus.

Example 17 Plasminogen Lowers Low-Density Lipoprotein Cholesterol inSerum of Diabetic Mice

Ten 24- to 25-week-old male db/db mice were randomly grouped, 5 mice ineach of the group administered with plasminogen and the control groupadministered with vehicle PBS. Three db/m mice were taken as the normalcontrol group. The mice were weighed and grouped on the day when theexperiment began, i.e. Day 0. Plasminogen or PBS was administered fromday 1 for 31 consecutive days. Mice in the group administered withplasminogen were injected with human plasminogen at a dose of 2 mg/0.2mL/mouse/day via the tail vein, an equal volume of PBS was administeredto mice in the PBS control group via the tail vein, and mice in thenormal control group received no treatment. On Day 32, the whole bloodwas collected from removed eyeballs in mice, and centrifuged at 3500r/min at 4° C. for 10 min to obtain a supernatant, which was detectedfor the concentration of low-density lipoprotein cholesterol (LDL-C) inserum using a low-density lipoprotein cholesterol detection kit (NanjingJiancheng Bioengineering Institute, Cat# A113-1).

The results showed that after continuous injection of human plasminogeninto diabetic model mice for 31 days, the content of LDL-C in serum ofmice in the group administered with plasminogen was lower than that inthe control group administered with vehicle PBS, and the statisticaldifference was close to significant (P=0.1) (FIG. 17). It indicates thatplasminogen can lower the content of LDL-C in serum.

Example 18 Plasminogen Lowers the Content of Serum Total Cholesterol inApoE Atherosclerosis Mice

Thirteen 6-week-old male ApoE mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([40,41]). The model mice continued to be fedwith a high-fat and high-cholesterol diet. 50 μL of blood was taken fromeach mouse three days before administration, and the total cholesterol(T-CHO) content was detected. The mice were randomly divided into twogroups based on the T-CHO content, 7 mice in the control groupadministered with vehicle PBS, and 6 mice in the group administered withplasminogen. The first day of administration was set as Day 1. Mice inthe group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein, both lasting for 30days. On Day 30, the mice fasted for 16 hours, and on Day 31, the bloodwas collected from removed eyeballs, and centrifuged to obtain asupernatant, which was detected for the total cholesterol using a totalcholesterol detection kit (Nanjing Jiancheng Bioengineering Institute,Cat# A111-1).

The detection results showed that the concentration of total cholesterolin mice in the group administered with plasminogen was remarkably lowerthan that in the control group administered with vehicle PBS, and thestatistical difference was significant (P=0.014) (FIG. 18). It indicatesthat plasminogen can lower the content of total cholesterol in serum ofApoE atherosclerosis model mice, and improve the dyslipidemia ofatherosclerosis.

Example 19 Plasminogen Lowers the Content of Serum Triglyceride in ApoEAtherosclerosis Mice

Thirteen 6-week-old male ApoE mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([40,41]). The model mice continued to be fedwith a high-fat and high-cholesterol diet. 50 μL of blood was taken fromeach mouse three days before administration, and the total cholesterol(T-CHO) content was detected. The mice were randomly divided into twogroups based on the T-CHO content, 7 mice in the control groupadministered with vehicle PBS, and 6 mice in the group administered withplasminogen. The first day of administration was recorded as Day 1. Micein the group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein, both lasting for 30days. On Day 30, the mice fasted for 16 hours, and on Day 31, the bloodwas collected from removed eyeballs, and centrifuged to obtain asupernatant, which was detected for triglyceride using a triglyceridedetection kit (Nanjing Jiancheng Bioengineering Institute, Cat# A110-1).

The detection results showed that the concentration of triglyceride inmice in the group administered with plasminogen was remarkably lowerthan that in the control group administered with vehicle PBS, and thestatistical difference was significant (P=0.013) (FIG. 19). It indicatesthat plasminogen can lower the content of triglyceride in serum of ApoEatherosclerosis model mice, and improve the dyslipidemia ofatherosclerosis.

Example 20 Plasminogen Lowers the Content of Serum Low-DensityLipoprotein Cholesterol in ApoE Atherosclerosis Mice

Thirteen 6-week-old male ApoE mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([40,41]). The model mice continued to be fedwith a high-fat and high-cholesterol diet. 50 μL of blood was taken fromeach mouse three days before administration, and the total cholesterol(T-CHO) content was detected. The mice were randomly divided into twogroups based on the T-CHO content, 7 mice in the control groupadministered with vehicle PBS, and 6 mice in the group administered withplasminogen. The first day of administration was recorded as Day 1. Micein the group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein, both lasting for 30days. On Day 30, the mice fasted for 16 hours, and on Day 31, the bloodwas collected from removed eyeballs, and centrifuged to obtain asupernatant, which was detected for LDL-C using a low-densitylipoprotein cholesterol (LDL-C) detection kit (Nanjing JianchengBioengineering Institute, Cat# A113-1).

The results showed that the concentration of LDL-C in mice in the groupadministered with plasminogen was remarkably lower than that in thecontrol group administered with vehicle PBS, and the statisticaldifference was significant (P=0.017) (FIG. 20). It indicates thatplasminogen can lower the content of low-density lipoprotein cholesterolin serum of ApoE atherosclerosis model mice, and improve thedyslipidemia in atherosclerosis model mice.

Example 21 Plasminogen Ameliorates Lipid Deposition in Liver of ApoEAtherosclerosis Mice

Thirteen 6-week-old male ApoE mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([40,41]). The model mice continued to be fedwith a high-fat and high-cholesterol diet. 50 μL of blood was taken fromeach mouse three days before administration, and the total cholesterol(T-CHO) content was detected. The mice were randomly divided into twogroups based on the T-CHO content, 7 mice in the control groupadministered with vehicle PBS, and 6 mice in the group administered withplasminogen. The first day of administration was recorded as Day 1. Micein the group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein, both lasting for 30days. The mice were sacrificed on Day 31. The liver tissues were fixedin 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and30% sucrose at 4° C. overnight, respectively, and embedded in OCT. Thefrozen sections were 8 μm thick, stained with oil red 0 for 15 min,differentiated with 75% ethanol for 5 s, followed by nuclear stainingwith hematoxylin for 30 s, and sealing with glycerine and gelatin. Thesections were observed under an optical microscope at 400×.

The staining results showed that the fat deposition in liver of mice inthe group administered with plasminogen (FIG. 21B) was remarkably lowerthan that in the control group administered with vehicle PBS (FIG. 21A),and the quantitative analysis showed significant statistical difference(P=0.02) (FIG. 21C). It indicates that plasminogen can reduce fatdeposition in liver of atherosclerosis model mice.

Example 22 Plasminogen Ameliorates Lipid Deposition in Aortic Sinus ofApoE Atherosclerosis Mice

Thirteen 6-week-old male ApoE mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([40,41]). The model mice continued to be fedwith a high-fat and high-cholesterol diet. 50 μL of blood was taken fromeach mouse three days before administration, and the total cholesterol(T-CHO) content was detected. The mice were randomly divided into twogroups based on the T-CHO content, 7 mice in the control groupadministered with vehicle PBS, and 6 mice in the group administered withplasminogen. The first day of administration was recorded as Day 1. Micein the group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein, both lasting for 30days. The mice were sacrificed on Day 31. The heart tissues were fixedin 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and30% sucrose at 4° C. overnight, respectively, and embedded in OCT. Thefrozen sections of aortic sinus were 8 jam thick, stained with oil red 0for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclearstaining with hematoxylin for 30 s, and sealing with glycerine andgelatin. The sections were observed under an optical microscope at 40×.

The staining results showed that the fat deposition in aortic sinus ofmice in the group administered with plasminogen (FIG. 22B) wasremarkably lower than that in the control group administered withvehicle PBS (FIG. 22A). It indicates that plasminogen can reduce lipiddeposition in aortic sinus of atherosclerosis model mice.

Example 23 Plasminogen Reduces Aortic Sinus Fibrosis in 16-WeekHyperlipemia Model Mice

Eleven 6-week-old male C57 mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([30,31]). This model was designated as the16-week hyperlipemia model. The model mice continued to be fed with ahigh-cholesterol diet. 50 μL of blood was taken from each mouse threedays before administration, and the total cholesterol (T-CHO) contentwas detected. The mice were randomly divided into two groups based onthe T-CHO content, 6 mice in the control group administered with vehiclePBS, and 5 mice in the group administered with plasminogen. The firstday of administration was recorded as Day 1. Mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume ofPBS was administered to mice in the control group administered withvehicle PBS via the tail vein. The mice were administered for 30 daysand sacrificed on Day 31. The hearts were fixed in 4% paraformaldehydefor 24 to 48 hours. The fixed tissues were paraffin-embedded afterdehydration with alcohol gradient and permeabilization with xylene. Theaortic sinus sections was 3 μm thick. The sections were dewaxed andrehydrated and washed with water once. After stained with 0.1% Siriusred in saturated picric acid for 30 min, the sections were flushed withrunning water for 2 min. After stained with hematoxylin for 1 min, thesections were flushed with running water, differentiated with 1%hydrochloric acid in alcohol, returned to blue with ammonia water,flushed with running water, dried and sealed with a neutral gum. Thesections were observed under an optical microscope at 40× (FIGS. 23A and23B) and 200× (FIGS. 23C and 23D).

The results showed that the area of collagen deposition (indicated byarrow) on the inner walls of blood vessels of aortic sinus in the groupadministered with plasminogen (FIGS. 23B and 23D) was remarkably lessthan that in the control group administered with vehicle PBS (FIGS. 23Aand 23C), indicating that plasminogen can alleviate the level of aorticsinus fibrosis in hyperlipemia model mice.

Example 24 Plasminogen Ameliorates Compensatory Cardiac Hypertrophy inApoE Atherosclerosis Mice

Thirteen 6-week-old male ApoE mice were fed with a high-fat andhigh-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to inducethe hyperlipemia model^([40,41]). 50 μL of blood was taken from eachmodel mouse three days before administration, and the total cholesterol(T-CHO) content was detected. The mice were randomly divided into twogroups based on the T-CHO content, 7 mice in the control groupadministered with vehicle PBS, and 6 mice in the group administered withplasminogen. The first day of administration was set as Day 1. Mice inthe group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein. The administrationlasted for 30 days. During the administration, mice continued to be fedwith a high-fat and high-cholesterol diet. After weighed on Day 31 ofadministration, the mice were sacrificed, their hearts were weighed, andcardiac coefficients were calculated. Cardiac coefficient (%)=heartweight/body weight×100.

The results showed that the cardiac coefficient of mice in the groupadministered with plasminogen was remarkably lower than that in thecontrol group administered with vehicle PBS (FIG. 24). It indicates thatplasminogen can alleviate the compensatory cardiac hypertrophy caused bycardiac injury in ApoE atherosclerosis model mice.

Example 25 Plasminogen Lowers Renal Fibrosis in 3% CholesterolHyperlipemia Model Mice

Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fatdiet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia^([30,31]).This model was designated as the 3% cholesterol hyperlipemia model. Themodel mice continued to be fed with the 3% cholesterol high-fat diet.Another five male C57 mice of the same week age were taken as the blankcontrol group, and were fed with a normal maintenance diet during theexperiment. 50 μL of blood was taken from each mouse three days beforeadministration, and the total cholesterol was detected. The model micewere randomly divided into two groups based on the total cholesterolconcentration and the body weight, i.e., the group administered withplasminogen, and the control group administered with vehicle PBS, 8 micein each group. The first day of administration was recorded as Day 1.Mice in the group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein. The mice wereadministered for 30 days. After the mice were administered on day 30,the mice were sacrificed on Day 31. The kidneys were fixed in 4%paraformaldehyde for 24 to 48 hours. The fixed tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The sections was 3 μm thick. The sectionswere dewaxed and rehydrated and washed with water once. After stainedwith 0.1% Sirius red in saturated picric acid for 30 min, the sectionswere flushed with running water for 2 min After stained with hematoxylinfor 1 min, the sections were flushed with running water, differentiatedwith 1% hydrochloric acid in alcohol, returned to blue with ammoniawater, flushed with running water, dried and sealed with a neutral gum.The sections were observed under an optical microscope at 200×.

The results showed that the collagen deposition in kidney (indicated byarrow) in the group administered with plasminogen (FIG. 25C) wasremarkably less than that in the control group administered with vehiclePBS (FIG. 25B), and the statistical difference was significant (FIG.25D); while in the group administered with plasminogen, fibrosis wassubstantially restored to a normal level (FIG. 25A). It indicates thatplasminogen can effectively reduce renal fibrosis in 3% cholesterolhyperlipemia model mice.

Example 26 Plasminogen Lowers Fat Deposition in Kidney of 3% CholesterolHyperlipemia Model Mice

Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fatdiet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia^([30,31]).This model was designated as the 3% cholesterol hyperlipemia model. Themodel mice continued to be fed with the 3% cholesterol high-fat diet.Another five male C57 mice of the same week age were taken as the blankcontrol group, and were fed with a normal maintenance diet during theexperiment. 50 μL of blood was taken from each mouse three days beforeadministration, and the total cholesterol was detected. The model micewere randomly divided into two groups based on the total cholesterolconcentration and the body weight, i.e., the group administered withplasminogen, and the control group administered with vehicle PBS, 8 micein each group. The first day of administration was recorded as Day 1.Mice in the group administered with plasminogen were injected with humanplasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and anequal volume of PBS was administered to mice in the control groupadministered with vehicle PBS via the tail vein, both lasting for 30days. The mice were sacrificed on Day 31. The kidneys were fixed in 4%paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30%sucrose at 4° C. overnight, respectively, and embedded in OCT. Thefrozen sections were 8 μm thick, stained with oil red 0 for 15 min,differentiated with 75% ethanol for 5 s, followed by nuclear stainingwith hematoxylin for 30 s, and sealing with glycerine and gelatin. Thesections were observed under an optical microscope at 400×.

The results showed that the fat deposition in kidney (indicated byarrow) of mice in the group administered with plasminogen (FIG. 26C) wasremarkably less than that in the control group administered with vehiclePBS (FIG. 26B), and the quantitative analysis showed significantstatistical difference (FIG. 26D); in addition, the lipid depositionlevel in the group administered with plasminogen was similar to that inmice in the blank control group (FIG. 26A). It indicates thatplasminogen can reduce the fat deposition in kidney of hyperlipemiamodel mice, and thus reduce a renal injury caused by fat deposition.

REFERENCES

-   [1] Edited by Zhao Kejian. Manual of Modern Pharmic Terms. Beijing:    China Medical Science Press. 2004. page 533-   [2] Shafrir E., Raz. I. Diabetes: Mellitus or Lipidus [J].    Diabetologia, 2003, 46:433-440.-   [3] Mooradian A D. Cardiovascular disease in type 2 diabetes    mellitus: Current management guidelines [J]. Arch Intern Med. 2003,    163:33-40.-   [4] Appel G. Lipid adnormalities in renal disease [J]. Kidney Int,    1991, 39:169.-   [5] J Zah, Kov, H Varerkov, et al., Dislipoproteinemia and Chronic    Kidney Failure [J]. VnitrLek. 2000, 46(9):539-46.-   [6] EffatRazeghi, Mohammadreza Shafipour et al. Lipid Disturbances    Before and After Renal Transplant. EffatRazeghi et al/Experimental    and Clinical Transplantation (2011) 4: 230-235-   [7] Grone H J. Glomerular lipids in non-hereditary forms of    glomerulopathy/glomerulo nephritis [J]. Nephrol. Dial Transplant.    1999, 14:1595-1598.-   [8] Larking R C. Dunlop M E, The link between hyperglycaemia and    diabetic nephropathy [J] Athrosclerosis, 2001, 156(2):25-33.-   [9] Alexander C M and Werb, Z. (1991). Extracellular matrix    degradation. In Cell Biology of Extracellular Matrix, Hay E D, ed.    (New York: Plenum Press), pp. 255-302.-   [10] Werb, Z., Mainardi, C. L., Vater, C. A., and Harris, E. D., Jr.    (1977). Endogenous activiation of latent collagenase by rheumatoid    synovial cells. Evidence for a role of plasminogen activator. N.    Engl. J. Med. 296, 1017-1023.-   [11] He, C. S., Wilhelm, S. M., Pentland, A. P., Marmer, B. L.,    Grant, G. A., Eisen, A. Z., and Goldberg, G. I. (1989). Tissue    cooperation in a proteolytic cascade activating human interstitial    collagenase. Proc. Natl. Acad. Sci. U. S. A 86, 2632-2636.-   [12] Stoppelli, M. P., Corti, A., Soffientini, A., Cassani, G.,    Blasi, F., and Assoian, R. K. (1985). Differentiation-enhanced    binding of the amino-terminal fragment of human urokinase    plasminogen activator to a specific receptor on U937 monocytes.    Proc. Natl. Acad. Sci. U. S. A 82, 4939-4943.-   [13] Vassalli, J. D., Baccino, D., and Belin, D. (1985). A cellular    binding site for the Mr 55,000 form of the human plasminogen    activator, urokinase. J. Cell Biol. 100, 86-92.-   [14] Wiman, B. and Wallen, P. (1975). Structural relationship    between “glutamic acid” and “lysine” forms of human plasminogen and    their interaction with the NH2-terminal activation peptide as    studied by affinity chromatography. Eur. J. Biochem. 50, 489-494.-   [15] Saksela, O. and Rifkin, D. B. (1988). Cell-associated    plasminogen activation: regulation and physiological functions.    Annu. Rev. Cell Biol. 4, 93-126.-   [16] Raum, D., Marcus, D., Alper, C. A., Levey, R., Taylor, P. D.,    and Starzl, T. E. (1980). Synthesis of human plasminogen by the    liver. Science 208, 1036-1037.-   [17] Wallén P (1980). Biochemistry of plasminogen. In Fibrinolysis,    Kline D L and Reddy K K N, eds. (Florida: CRC)-   [18] Sottrup-Jensen, L., Zajdel, M., Claeys, H., Petersen, T. E.,    and Magnusson, S. (1975). Amino-acid sequence of activation cleavage    site in plasminogen: homology with “pro” part of prothrombin. Proc.    Natl. Acad. Sci. U. S. A 72, 2577-2581.-   [19] Collen, D. and Lijnen, H. R. (1991). Basic and clinical aspects    of fibrinolysis and thrombolysis. Blood 78, 3114-3124.-   [20] Alexander, C. M. and Werb, Z. (1989). Proteinases and    extracellular matrix remodeling. Curr. Opin. Cell Biol. 1, 974-982.-   [21] Mignatti, P. and Rifkin, D. B. (1993). Biology and biochemistry    of proteinases in tumor invasion. Physiol Rev. 73, 161-195.-   [22] Collen, D. (2001). Ham-Wasserman lecture: role of the    plasminogen system in fibrin-homeostasis and tissue remodeling.    Hematology. (Am. Soc. Hematol. Educ. Program.) 1-9.-   [23] Rifkin, D. B., Moscatelli, D., Bizik, J., Quarto, N., Blei, F.,    Dennis, P., Flaumenhaft, R., and Mignatti, P. (1990). Growth factor    control of extracellular proteolysis. Cell Differ. Dev. 32, 313-318.-   [24] Andreasen, P. A., Kjoller, L., Christensen, L., and    Duffy, M. J. (1997). The urokinase-type plasminogen activator system    in cancer metastasis: a review. Int. J. Cancer 72, 1-22.-   [25] Rifkin, D. B., Mazzieri, R., Munger, J. S., Noguera, I., and    Sung, J. (1999). Proteolytic control of growth factor availability.    APMIS 107, 80-85.-   [26] Marder V J, Novokhatny V. Direct fibrinolytic agents:    biochemical attributes, preclinical foundation and clinical    potential [J]. Journal of Thrombosis and Haemostasis, 2010, 8(3):    433-444.-   [27] Hunt J A, Petteway Jr S R, Scuderi P, et al. Simplified    recombinant plasmin production and functional comparison of a novel    thrombolytic molecule with plasma-derived plasmin [J].    ThrombHaemost, 2008, 100(3): 413-419.-   [28] Sottrup-Jensen L, Claeys H, Zajdel M, et al. The primary    structure of human plasminogen: Isolation of two lysine-binding    fragments and one “mini”-plasminogen (MW, 38,000) by    elastase-catalyzed-specific limited proteolysis [J]. Progress in    chemical fibrinolysis and thrombolysis, 1978, 3: 191-209.-   [29] Nagai N, Demarsin E, Van Hoef B, et al. Recombinant human    microplasmin: production and potential therapeutic properties [J].    Journal of Thrombosis and Haemostasis, 2003, 1(2): 307-313.-   [30] Dominika Nackiewicz, Paromita Dey, Barbara Szczerba et al    Inhibitor of differentiation 3, a transcription factor regulates    hyperlipidemia associated kidney disease. Nephron Exp Nephrol. 2014;    126(3): 141-147.-   [31] Ming Gul, Yu Zhang., Shengjie Fan et al. Extracts of    RhizomaPolygonatiOdorati Prevent High-Fat Diet-Induced Metabolic    Disorders in C57BL/6 Mice. PLoS ONE 8(11): e81724.-   [32] Siobhan M. Craige, PhD, Shashi Kant et al. Endothelial NADPH    oxidase 4 protects ApoE−/− mice from atherosclerotic lesions. Free    RadicBiol Med. 2015 December; 89: 1-7.-   [33] Dimitrios Davalos, Katerina Akassoglou. Fibrinogen as a key    regulator of inflammation in disease. Seminars in    Immunopathology, 2012. 34(1):43-62.-   [34] Valvi D, Mannino D M, Mullerova H, et al. Fibrinogen, chronic    obstructive pulmonary disease (COPD) and outcomes in two United    States cohorts. Int J Chron Obstruct Pulmon Dis 2012; 7:173-82.-   [35] Zhang M, Takahashi K, Alicot E M, Vorup-Jensen T, Kessler B, et    al. (2006) Activation of the lectin pathway by natural IgM in a    model of ischemia reperfusion injury. J Immunol 177: 4727-4734.-   [36] Kim S J, Gershov D, Ma X, Brot N, Elkon K B (2002) I-PLA2    Activation during Apoptosis Promotes the Exposure of Membrane    Lysophosphatidylcholine Leading to Binding by Natural Immunoglobulin    M Antibodies and Complement Activation. The Journal of Experimental    Medicine 196: 655-665.-   [37] R. Langhorn and J. L. Willesen. Cardiac Troponins in Dogs and    Cats. J Vet Intern Med 2016; 30:36-50.-   [38] Sungwon Lee, Youngjoo Lee, Jiyeon Kim et al. Atorvastatin and    rosuvastatin improve physiological parameters and alleviate immune    dysfunction in metabolic disorders. Biochem Biophys Res Commun. 2016    Sep. 23; 478(3):1242-7.-   [39] Ametov A S, Kulidzhanian N K. Diabetes mellitus is an    independent risk factor for cardiovascular disease. Ter Arkh. 2012;    84(8):91-4.-   [40] Yutaka Nakashima, Andrew S. Plump, Elaine W. Raines et al.    Arterioscler Thromb. 1994 January; 14(1):133-40.-   [41] Yvonne Nitschke, Gabriele Weissen-Plenz, Robert Terkeltaub et    al. Nppl promotes atherosclerosis in ApoE knockout mice. J. Cell.    Mol. Med. Vol 15, No 11, 2011 pp. 2273-2283.

1: A method for preventing and/or treating a fat metabolism disorder andits related conditions in a subject, comprising administering aprophylactically and/or therapeutically effective amount of plasminogento the subject, wherein the subject is susceptible to a fat metabolismdisorder, suffers from a fat metabolism disorder or other diseasesaccompanied by a fat metabolism disorder. 2: The method of claim 1,wherein the fat metabolism disorder is a fat metabolism disorderelicited or accompanied by an endocrine disorder disease, a glucosemetabolism disease, a liver disease, a kidney disease, a cardiovasculardisease, an intestinal disease, a thyroid disease, a gallbladder or abiliary tract disease, obesity, drinking, and a drug therapy. 3: Themethod of claim 2, wherein the fat metabolism disorder is a fatmetabolism disorder elicited or accompanied by hypertension, diabetesmellitus, chronic hepatitis, hepatic cirrhosis, renal injury, chronicglomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renalinsufficiency, kidney transplantation, uremia, hypothyroidism,obstructive cholecystitis, obstructive cholangitis, and a drug orhormone therapy. 4: The method of claim 1, wherein the fat metabolismdisorder is hyperlipemia, hyperlipoproteinemia, fatty liver,atherosclerosis, obesity, and a visceral fat deposition. 5: The methodof claim 4, wherein the atherosclerosis comprises aorticatherosclerosis, coronary atherosclerosis, cerebral atherosclerosis,renal atherosclerosis, hepatic atherosclerosis, mesentericatherosclerosis, and lower limb atherosclerosis. 6: A method forpreventing and/or reducing an abnormal fat deposition in a body tissueand an organ of a subject, comprising administering an effective amountof plasminogen to the subject.
 7. (canceled) 8: The method of claim 6,wherein the abnormal fat deposition in a body tissue and an organ refersto an abnormal fat deposition in blood, a subcutaneous tissue, avascular wall, and an internal organ. 9: The method of claim 8, whereinthe condition resulting from the abnormal fat deposition in a bodytissue and an organ comprises obesity, hyperlipemia,hyperlipoproteinemia, fatty liver, atherosclerosis, a lipid-inducedcardiac damage, a lipid-induced renal damage, and a lipid-induced isletdamage. 10-15. (canceled) 16: A method for ameliorating hyperlipemia,preventing and/or treating a hyperlipemia-related condition in asubject, comprising administering an effective amount of plasminogen tothe subject. 17: The method of claim 16, wherein the hyperlipemia isselected from one or more of: hypercholesterolemia,hypertriglyceridemia, combined hyperlipemia, and hypo-high-densitylipoproteinemia. 18-22. (canceled) 23: The method of claim 16, whereinthe hyperlipemia-related condition comprises diabetes mellitus,hypertension, atherosclerosis, coronary heart disease, angina pectoris,myocardial infarction, arrhythmia, chronic hepatitis, fatty liver,hepatic cirrhosis, cerebral circulation insufficiency, cerebralischemia, cerebral infarction, chronic nephritis, chronicpyelonephritis, renal insufficiency, nephrotic syndrome, uremia, andobesity. 24: The method of claim 1, wherein the plasminogen isadministered in combination with one or more other drugs or therapies.25: The method of claim 24, wherein the one or more other drugscomprises a hypolipidemic drug, an anti-platelet drug, anantihypertensive drug, a vasodilator, a hypoglycemic drug, ananticoagulant drug, a thrombolytic drug, a hepatoprotective drug, ananti-arrhythmia drug, a cardiotonic drug, a diuretic drug, ananti-infective drug, an antiviral drug, an immunomodulatory drug, aninflammatory regulatory drug, an anti-tumor drug, a hormone drug, andthyroxine.
 26. (canceled) 27: The method of claim 25, wherein the drugscomprise hypolipidemic drugs: statins; fibrates; niacin; cholestyramine;clofibrate; unsaturated fatty acids such as Yishouning, Xuezhiping, andXinmaile; and alginic sodium diester; anti-platelet drugs: aspirin;dipyridamole; clopidogrel; and cilostazol; vasodilators: hydralazine;nitroglycerin, and isosorbide dinitrate; sodium nitroprusside;α1-receptor blockers such as prazosin; α-receptor blockers such asphentolamine; β2-receptor stimulants such as salbutamol; captopril,enalapril; nifedipine, diltiazem; and salbutamol, loniten,prostaglandin, and atrial natriuretic peptide; thrombolytic drugs:urokinase, and streptokinase; tissue-type plasminogen activators; singlechain urokinase-type plasminogen activators; and a TNK tissue-typeplasminogen activator; and anticoagulant drugs: heparin; enoxaparin;nadroparin; and bivalirudin. 28: The method of claim 1, wherein theplasminogen has at least 75% sequence identity with SEQ ID No. 2, andstill has the plasminogen activity.
 29. (canceled) 30: The method ofclaim 1, wherein the plasminogen is a protein that comprises aplasminogen active fragment and still has the plasminogen activity. 31:The method of claim 1, wherein the plasminogen is selected fromGlu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen,delta-plasminogen or their variants that retain the plasminogenactivity. 32: The method of claim 1, wherein the plasminogen is anatural or synthetic human plasminogen, or a variant or fragment thereofthat still retains the plasminogen activity. 33-46. (canceled) 47: Themethod of claim 1, wherein the plasminogen is administered to thesubject at a dosage of 1-100 mg/kg at a frequency of weekly to daily.48: The method of claim 47, wherein the dosage of the plasminogen isrepeated at least once. 49: The method of claim 47, wherein theplasminogen is administered at least daily.